JP7411175B2 - Lipocalin-type prostaglandin D2 synthase production promoter - Google Patents
Lipocalin-type prostaglandin D2 synthase production promoter Download PDFInfo
- Publication number
- JP7411175B2 JP7411175B2 JP2019190666A JP2019190666A JP7411175B2 JP 7411175 B2 JP7411175 B2 JP 7411175B2 JP 2019190666 A JP2019190666 A JP 2019190666A JP 2019190666 A JP2019190666 A JP 2019190666A JP 7411175 B2 JP7411175 B2 JP 7411175B2
- Authority
- JP
- Japan
- Prior art keywords
- pgds
- extract
- brain
- lipocalin
- synthase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 65
- 102000048176 Prostaglandin-D synthases Human genes 0.000 title claims description 43
- 108030003866 Prostaglandin-D synthases Proteins 0.000 title claims description 43
- 239000000284 extract Substances 0.000 claims description 77
- 210000003668 pericyte Anatomy 0.000 claims description 44
- 210000001519 tissue Anatomy 0.000 claims description 38
- 230000001737 promoting effect Effects 0.000 claims description 29
- 238000002360 preparation method Methods 0.000 claims description 28
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 19
- 241000700618 Vaccinia virus Species 0.000 claims description 15
- 208000028867 ischemia Diseases 0.000 claims description 11
- 239000004480 active ingredient Substances 0.000 claims description 8
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- 101710145576 Prostaglandin-H2 D-isomerase Proteins 0.000 description 145
- 102100033279 Prostaglandin-H2 D-isomerase Human genes 0.000 description 137
- 238000012360 testing method Methods 0.000 description 95
- 239000000126 substance Substances 0.000 description 76
- 210000004556 brain Anatomy 0.000 description 74
- 230000014509 gene expression Effects 0.000 description 45
- 238000000034 method Methods 0.000 description 45
- 230000000694 effects Effects 0.000 description 27
- 108090000623 proteins and genes Proteins 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 26
- BHMBVRSPMRCCGG-OUTUXVNYSA-N prostaglandin D2 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](C\C=C/CCCC(O)=O)[C@@H](O)CC1=O BHMBVRSPMRCCGG-OUTUXVNYSA-N 0.000 description 25
- BHMBVRSPMRCCGG-UHFFFAOYSA-N prostaglandine D2 Natural products CCCCCC(O)C=CC1C(CC=CCCCC(O)=O)C(O)CC1=O BHMBVRSPMRCCGG-UHFFFAOYSA-N 0.000 description 25
- 206010012289 Dementia Diseases 0.000 description 20
- 238000009826 distribution Methods 0.000 description 20
- 239000003814 drug Substances 0.000 description 20
- 102000004169 proteins and genes Human genes 0.000 description 19
- 241000699670 Mus sp. Species 0.000 description 18
- 208000026106 cerebrovascular disease Diseases 0.000 description 18
- 238000011156 evaluation Methods 0.000 description 18
- 206010008118 cerebral infarction Diseases 0.000 description 17
- 210000003491 skin Anatomy 0.000 description 17
- 229940079593 drug Drugs 0.000 description 16
- 238000011282 treatment Methods 0.000 description 16
- 210000003556 vascular endothelial cell Anatomy 0.000 description 16
- 206010046865 Vaccinia virus infection Diseases 0.000 description 14
- 239000003463 adsorbent Substances 0.000 description 14
- 208000007089 vaccinia Diseases 0.000 description 14
- 230000002265 prevention Effects 0.000 description 13
- 102000019298 Lipocalin Human genes 0.000 description 12
- 108050006654 Lipocalin Proteins 0.000 description 12
- 238000000605 extraction Methods 0.000 description 12
- 230000001965 increasing effect Effects 0.000 description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 230000006870 function Effects 0.000 description 11
- 230000000302 ischemic effect Effects 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 208000024827 Alzheimer disease Diseases 0.000 description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 238000011532 immunohistochemical staining Methods 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 9
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 8
- 108010088225 Nestin Proteins 0.000 description 8
- 102000008730 Nestin Human genes 0.000 description 8
- 239000002033 PVDF binder Substances 0.000 description 8
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 8
- 230000003431 anti-prostaglandin Effects 0.000 description 8
- 239000002771 cell marker Substances 0.000 description 8
- 239000012228 culture supernatant Substances 0.000 description 8
- 239000008103 glucose Substances 0.000 description 8
- 210000005055 nestin Anatomy 0.000 description 8
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 8
- 238000003757 reverse transcription PCR Methods 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 230000009471 action Effects 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 7
- 210000004379 membrane Anatomy 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- YIBNHAJFJUQSRA-YNNPMVKQSA-N prostaglandin H2 Chemical compound C1[C@@H]2OO[C@H]1[C@H](/C=C/[C@@H](O)CCCCC)[C@H]2C\C=C/CCCC(O)=O YIBNHAJFJUQSRA-YNNPMVKQSA-N 0.000 description 7
- 239000011251 protective drug Substances 0.000 description 7
- 230000001681 protective effect Effects 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- VHRUMKCAEVRUBK-WKELIDJCSA-N (z)-7-[(1s,5e)-5-[(z)-oct-2-enylidene]-4-oxocyclopent-2-en-1-yl]hept-5-enoic acid Chemical compound CCCCC\C=C/C=C1\[C@@H](C\C=C/CCCC(O)=O)C=CC1=O VHRUMKCAEVRUBK-WKELIDJCSA-N 0.000 description 6
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 230000003930 cognitive ability Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 231100000673 dose–response relationship Toxicity 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 230000000147 hypnotic effect Effects 0.000 description 6
- 210000002569 neuron Anatomy 0.000 description 6
- 230000000144 pharmacologic effect Effects 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 235000002639 sodium chloride Nutrition 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 102000007469 Actins Human genes 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 5
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 230000002055 immunohistochemical effect Effects 0.000 description 5
- 238000000386 microscopy Methods 0.000 description 5
- 210000005036 nerve Anatomy 0.000 description 5
- 150000003180 prostaglandins Chemical class 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- VSRXYLYXIXYEST-KZTWKYQFSA-N 13,14-dihydro-15-ketoprostaglandin D2 Chemical compound CCCCCC(=O)CC[C@@H]1[C@@H](C\C=C/CCCC(O)=O)[C@@H](O)CC1=O VSRXYLYXIXYEST-KZTWKYQFSA-N 0.000 description 4
- IGAZHQIYONOHQN-UHFFFAOYSA-N Alexa Fluor 555 Chemical compound C=12C=CC(=N)C(S(O)(=O)=O)=C2OC2=C(S(O)(=O)=O)C(N)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C(O)=O IGAZHQIYONOHQN-UHFFFAOYSA-N 0.000 description 4
- 208000016192 Demyelinating disease Diseases 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- 230000006933 amyloid-beta aggregation Effects 0.000 description 4
- 210000003710 cerebral cortex Anatomy 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 4
- 229960002986 dinoprostone Drugs 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- -1 magnesium chloride Chemical class 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 230000003449 preventive effect Effects 0.000 description 4
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 4
- UQOQENZZLBSFKO-POPPZSFYSA-N prostaglandin J2 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](C\C=C/CCCC(O)=O)C=CC1=O UQOQENZZLBSFKO-POPPZSFYSA-N 0.000 description 4
- 239000002516 radical scavenger Substances 0.000 description 4
- KJDSORYAHBAGPP-UHFFFAOYSA-N 4-(3,4-diaminophenyl)benzene-1,2-diamine;hydron;tetrachloride Chemical compound Cl.Cl.Cl.Cl.C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 KJDSORYAHBAGPP-UHFFFAOYSA-N 0.000 description 3
- 206010012305 Demyelination Diseases 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 206010021143 Hypoxia Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000003542 behavioural effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000017531 blood circulation Effects 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 210000003169 central nervous system Anatomy 0.000 description 3
- 230000003920 cognitive function Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 239000012156 elution solvent Substances 0.000 description 3
- 238000010195 expression analysis Methods 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 206010022437 insomnia Diseases 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 210000003657 middle cerebral artery Anatomy 0.000 description 3
- 239000003068 molecular probe Substances 0.000 description 3
- 210000001178 neural stem cell Anatomy 0.000 description 3
- 238000012758 nuclear staining Methods 0.000 description 3
- 210000004248 oligodendroglia Anatomy 0.000 description 3
- 235000020030 perry Nutrition 0.000 description 3
- 239000006187 pill Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000004043 responsiveness Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 230000002792 vascular Effects 0.000 description 3
- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 2
- JQMFQLVAJGZSQS-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-N-(2-oxo-3H-1,3-benzoxazol-6-yl)acetamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)NC1=CC2=C(NC(O2)=O)C=C1 JQMFQLVAJGZSQS-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 239000012103 Alexa Fluor 488 Substances 0.000 description 2
- 239000005995 Aluminium silicate Substances 0.000 description 2
- 208000037259 Amyloid Plaque Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000008035 Back Pain Diseases 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 206010048962 Brain oedema Diseases 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 206010008089 Cerebral artery occlusion Diseases 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 102100029100 Hematopoietic prostaglandin D synthase Human genes 0.000 description 2
- 101710082112 Hematopoietic prostaglandin D synthase Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 208000008930 Low Back Pain Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 238000012347 Morris Water Maze Methods 0.000 description 2
- 206010029350 Neurotoxicity Diseases 0.000 description 2
- 101150020237 PTGDS gene Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 102000009389 Prostaglandin D receptors Human genes 0.000 description 2
- 108050000258 Prostaglandin D receptors Proteins 0.000 description 2
- 102100024212 Prostaglandin D2 receptor Human genes 0.000 description 2
- 239000012083 RIPA buffer Substances 0.000 description 2
- 238000011530 RNeasy Mini Kit Methods 0.000 description 2
- 206010044221 Toxic encephalopathy Diseases 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 235000012211 aluminium silicate Nutrition 0.000 description 2
- 230000000202 analgesic effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000000576 arachnoid Anatomy 0.000 description 2
- 210000001130 astrocyte Anatomy 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 208000006752 brain edema Diseases 0.000 description 2
- 208000029028 brain injury Diseases 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000517 effect on sleep Effects 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000007954 hypoxia Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 201000007309 middle cerebral artery infarction Diseases 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000023105 myelination Effects 0.000 description 2
- 208000004296 neuralgia Diseases 0.000 description 2
- 231100000228 neurotoxicity Toxicity 0.000 description 2
- 230000007135 neurotoxicity Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 210000000578 peripheral nerve Anatomy 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 210000003446 pia mater Anatomy 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 210000004116 schwann cell Anatomy 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 229940125724 sleeping drug Drugs 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- AWSUSADYFDCYML-KUPUPYBPSA-N (z)-7-[(1s,5r)-5-[(e)-oct-1-enyl]-4-oxocyclopent-2-en-1-yl]hept-5-enoic acid Chemical compound CCCCCC\C=C\[C@@H]1[C@@H](C\C=C/CCCC(O)=O)C=CC1=O AWSUSADYFDCYML-KUPUPYBPSA-N 0.000 description 1
- HMUNWXXNJPVALC-UHFFFAOYSA-N 1-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C(CN1CC2=C(CC1)NN=N2)=O HMUNWXXNJPVALC-UHFFFAOYSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- CONKBQPVFMXDOV-QHCPKHFHSA-N 6-[(5S)-5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-2-oxo-1,3-oxazolidin-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C[C@H]1CN(C(O1)=O)C1=CC2=C(NC(O2)=O)C=C1 CONKBQPVFMXDOV-QHCPKHFHSA-N 0.000 description 1
- 238000010175 APPswe/PSEN1dE9 Methods 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 101150043085 COCH gene Proteins 0.000 description 1
- 101000925430 Caenorhabditis elegans Sphingomyelin phosphodiesterase 1 Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 206010008120 Cerebral ischaemia Diseases 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 101100532034 Drosophila melanogaster RTase gene Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 101150092680 GBP6 gene Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 101001135402 Homo sapiens Prostaglandin-H2 D-isomerase Proteins 0.000 description 1
- 241001562081 Ikeda Species 0.000 description 1
- 208000028226 Krabbe disease Diseases 0.000 description 1
- 241000283953 Lagomorpha Species 0.000 description 1
- 241000283986 Lepus Species 0.000 description 1
- 241000025416 Lepus brachyurus Species 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000283965 Ochotona princeps Species 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000000114 Pain Threshold Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 206010034464 Periarthritis Diseases 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 101150034985 Ptgdr2 gene Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010039085 Rhinitis allergic Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 206010058009 Subacute myelo-opticoneuropathy Diseases 0.000 description 1
- 208000032851 Subarachnoid Hemorrhage Diseases 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 201000010105 allergic rhinitis Diseases 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000003376 axonal effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 230000006931 brain damage Effects 0.000 description 1
- 231100000874 brain damage Toxicity 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000002987 choroid plexus Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000003710 glymphatic flow Effects 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 239000003326 hypnotic agent Substances 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000037041 intracellular level Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 208000023589 ischemic disease Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 210000003140 lateral ventricle Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229910052762 osmium Inorganic materials 0.000 description 1
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 230000037040 pain threshold Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008447 perception Effects 0.000 description 1
- 230000003617 peroxidasic effect Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- KSSNXJHPEFVKHY-UHFFFAOYSA-N phenol;hydrate Chemical compound O.OC1=CC=CC=C1 KSSNXJHPEFVKHY-UHFFFAOYSA-N 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- CMDGQTVYVAKDNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrate Chemical compound O.OCC(O)CO CMDGQTVYVAKDNA-UHFFFAOYSA-N 0.000 description 1
- 150000003163 prostaglandin D2 derivatives Chemical class 0.000 description 1
- 150000003176 prostaglandin J2 derivatives Chemical class 0.000 description 1
- 230000009993 protective function Effects 0.000 description 1
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5073—Stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/20—Hypnotics; Sedatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/065—Poxviridae, e.g. avipoxvirus
- G01N2333/07—Vaccinia virus; Variola virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/99—Isomerases (5.)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2864—Sleep disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Dermatology (AREA)
- Hospice & Palliative Care (AREA)
- Psychiatry (AREA)
- Anesthesiology (AREA)
- Vascular Medicine (AREA)
Description
本発明は、リポカリン型プロスタグランジンD2合成酵素（以下「L-PGDS」と表記することがある。また、「リポカリン型プロスタグランジンD合成酵素」と称するものも本発明に包含され得る。）の産生促進剤等に関するものである。 The present invention relates to lipocalin-type prostaglandin D2 synthase (hereinafter sometimes referred to as "L-PGDS". Also, what is referred to as "lipocalin-type prostaglandin D synthase" may also be included in the present invention). This relates to production promoters, etc.
プロスタグランジンD2合成酵素（PGDS）には、中枢神経系、雄性生殖器、心臓等に分布するリポカリン型（L-）PGDSと、肥満細胞やTh2細胞に分布する造血器型（H-）PGDSの2種類があることが知られている。L-PGDSは、プロスタグランジン生合成の共通中間体であるプロスタグランジンH2（PGH2）からプロスタグランジンD2（PGD2）への異性化反応を触媒する活性を有する。他方、L-PGDSは、構造上は脂溶性物質のキャリアとして機能するリポカリンファミリーに属している。従って、L-PGDSは、PGD2生合成酵素と脂溶性キャリアの両面性を有する多機能タンパクである。 Prostaglandin D2 synthase (PGDS) includes lipocalin-type (L-) PGDS, which is distributed in the central nervous system, male reproductive organs, and heart, and hematopoietic-type (H-) PGDS, which is distributed in mast cells and Th2 cells. It is known that there are two types. L-PGDS has the activity of catalyzing the isomerization reaction from prostaglandin H2 (PGH2), which is a common intermediate in prostaglandin biosynthesis, to prostaglandin D2 (PGD2). On the other hand, L-PGDS structurally belongs to the lipocalin family, which functions as a carrier of fat-soluble substances. Therefore, L-PGDS is a multifunctional protein that functions as both a PGD2 biosynthetic enzyme and a fat-soluble carrier.
中枢神経系に分布するL-PGDSは脳脊髄液にも検出され、他のリポカリンと比較して巨大な親油性ポケットを有するため、脳内の様々な脂溶性分子の輸送タンパク質及びスカベンジャーとしての役割を担っていると考えられている。そのようなL-PGDSのリポカリンとしての機能の例としては、くも膜下出血後に脳内L-PGDSが上昇し、神経毒性の原因となるビリルビンを抱合することで神経毒性を回避させること（Inui T. et al., J. Cereb. Blood Flow Metab. 34, 1558-1567, 2014）や、L-PGDSがアルツハイマー病の患者や動物モデルにおいて、アミロイドβタンパク質（Aβ、老人斑）のオリゴマー形成に必要な領域に強固に結合し、脳脊髄液中でのAβ凝集形成と細胞傷害性を阻害すること（Kanekiyo T. et al., Proc. Natl. Acad. Sci. USA 104, 6412-6417, 2007）等が挙げられる。 L-PGDS, which is distributed in the central nervous system, is also detected in the cerebrospinal fluid and has a large lipophilic pocket compared to other lipocalins, so it plays a role as a transport protein and scavenger for various lipid-soluble molecules in the brain. It is thought that it is responsible for An example of the function of L-PGDS as a lipocalin is that L-PGDS increases in the brain after subarachnoid hemorrhage and avoids neurotoxicity by conjugating bilirubin, which causes neurotoxicity (Inui T et al., J. Cereb. Blood Flow Metab. 34, 1558-1567, 2014) and L-PGDS is required for oligomerization of amyloid-β protein (Aβ, senile plaques) in Alzheimer's disease patients and animal models. binding tightly to the cerebrospinal fluid and inhibiting Aβ aggregate formation and cytotoxicity (Kanekiyo T. et al., Proc. Natl. Acad. Sci. USA 104, 6412-6417, 2007) etc.
また、マウスの虚血脳においてPGD2の産生が顕著に増加し、L-PGDS及びH-PGDS、又はPGD2受容体DP1のノックアウトにより虚血後の脳浮腫発症が重症化するとの報告（Tanigichi H. et al., J. Neurosci. 27, 4303-4312, 2007）や、L-PGDSのノックアウトにより脳梗塞巣や脳浮腫が増大するとの報告（Saleem S. et al., Neuroscience 160, 248-254, 2009）がある。このことから、脳虚血時においてL-PGDSやH-PGDS により産生されるPGD2は、その受容体を介した作用により脳保護的に働くと考えられている。 In addition, it has been reported that the production of PGD2 significantly increases in the ischemic brain of mice, and that knockout of L-PGDS and H-PGDS or the PGD2 receptor DP1 aggravates the onset of post-ischemic cerebral edema (Tanigichi H. et al., J. Neurosci. 27, 4303-4312, 2007), and reports that knockout of L-PGDS increases cerebral infarct focus and cerebral edema (Saleem S. et al., Neuroscience 160, 248-254, 2009). From this, it is thought that PGD2 produced by L-PGDS and H-PGDS during cerebral ischemia acts in a brain-protective manner through its receptor-mediated action.
さらに、ガラクトシルセラミダーゼ欠損による脱髄性疾患クラベ病のモデルマウス（Twitcherマウス）では、脱髄に抵抗性を示す軸索領域でL-PGDS遺伝子発現の上昇が観察され、L-PGDSの欠損をさらに導入するとオリゴデンドロサイトの消失による脱髄の重症化が起こることが報告されている（Taniike M. et al., J. Neurosci. 22, 4885-4896, 2002）。また、L-PGDS欠損マウスでは、末梢神経の脱髄が誘導されると報告されており、シュワン細胞のPGD2受容体であるGpr44が神経髄鞘化に必要であることが見出されている（Trimarco A. et al., Nature Neurosci. 17, 1682-1992, 2014）。これらの知見から、オリゴデンドロサイト（中枢神経）やシュワン細胞（末梢神経）における神経軸索髄鞘化とその維持にL-PGDS及びその産物であるPGD2が必要であると考えられている。 Furthermore, in a model mouse (Twitcher mouse) of the demyelinating disease Clave disease caused by galactosylceramidase deficiency, increased L-PGDS gene expression was observed in axonal regions that are resistant to demyelination. It has been reported that when introduced, severe demyelination occurs due to disappearance of oligodendrocytes (Taniike M. et al., J. Neurosci. 22, 4885-4896, 2002). In addition, it has been reported that demyelination of peripheral nerves is induced in L-PGDS-deficient mice, and it has been found that Gpr44, a PGD2 receptor on Schwann cells, is required for nerve myelination ( Trimarco A. et al., Nature Neurosci. 17, 1682-1992, 2014). Based on these findings, it is believed that L-PGDS and its product PGD2 are required for nerve axon myelination and its maintenance in oligodendrocytes (central nerves) and Schwann cells (peripheral nerves).
なお、L-PGDSによる保護作用については、神経細胞に限らず、過酸化ストレスによる消化管グリア細胞の細胞死をPGD2の代謝産物である15-デオキシ-プロスタグランジンJ2が回避させることも報告されている（Abdo H. et al., J. Physiol. 590, 2739-2750, 2012）。 The protective effect of L-PGDS is not limited to nerve cells; it has also been reported that 15-deoxy-prostaglandin J2, a metabolite of PGD2, prevents cell death in gastrointestinal glial cells due to peroxidative stress. (Abdo H. et al., J. Physiol. 590, 2739-2750, 2012).
さらに、L-PGDSにより合成される脳内PGD2は睡眠調節作用を有することが古くから知られている（Ueno R. et al., Biochem. Biophys. Res. Commun. 109, 576-582, 1982）。その機序については、日本の研究者により詳細に検討された結果、PGD2が眼底部クモ膜DP1受容体を介してアデノシンを分泌することで睡眠中枢を刺激することによるものとされている。L-PGDSは酵素産物であるPGD2に対して高い親和性を有することから、脳脊髄液中でのPGD2の安定性を確保するとともにPGD2を近傍の受容体へ輸送すると推定されており（Urade Y. and Hayaishi O., Biochim. Biophys. Acta 1482, 259-271, 2000）、PGD2を介した睡眠調節に大きく関与すると考えられる。 Furthermore, it has been known for a long time that PGD2 in the brain, which is synthesized by L-PGDS, has a sleep regulating effect (Ueno R. et al., Biochem. Biophys. Res. Commun. 109, 576-582, 1982). . As for the mechanism, as a result of a detailed study by Japanese researchers, it is believed that PGD2 stimulates the sleep center by secreting adenosine via the arachnoid DP1 receptor in the fundus of the eye. Since L-PGDS has a high affinity for the enzyme product PGD2, it is assumed that it ensures the stability of PGD2 in the cerebrospinal fluid and transports PGD2 to nearby receptors (Urade Y and Hayaishi O., Biochim. Biophys. Acta 1482, 259-271, 2000), and is thought to be significantly involved in sleep regulation via PGD2.
上述したように、L-PGDSはPGD2を合成する酵素タンパク質として働くと共に、脳内の様々な疎水性低分子の結合・輸送及びスカベンジャーとしての役割を担い、脳内環境調整・脳保護機能や睡眠調節機能等の様々な機能を有するタンパク質であると考えられている。従って、L-PGDSの産生を促進することは、これらの機能を効果的に働かせることになり、L-PGDSが関連する疾患の予防・治療等に有用であると考えられる。しかしながら、これまでに生体内においてL-PGDSの産生を促進する物質の報告はなされていない。 As mentioned above, L-PGDS acts as an enzyme protein that synthesizes PGD2, and also plays a role in binding and transporting various hydrophobic low molecules in the brain and as a scavenger, and plays a role in regulating the brain environment, brain protection function, and sleep. It is thought to be a protein that has various functions such as regulatory functions. Therefore, promoting the production of L-PGDS makes these functions work effectively, and is considered to be useful for the prevention and treatment of diseases related to L-PGDS. However, no substance has been reported so far that promotes the production of L-PGDS in vivo.
本発明において、ワクシニアウイルス接種炎症組織抽出物（本抽出物）が優れたL-PGDS産生促進作用を有することが認められたが、本抽出物又はこれを含有する製剤については非常に多岐に及ぶ作用・効果が知られている。例えば、本抽出物の脳に対する作用・効果として、脳梗塞等の虚血性疾患に対する治療効果（特開２０００－１６９４２号公報）やBDNF等の神経栄養因子の産生促進作用（国際公開ＷＯ２０１１／１６２３１７号公報）が知られているが、本抽出物がL-PGDSの産生促進作用を有することはこれまで知られていない。 In the present invention, it has been recognized that the vaccinia virus-inoculated inflamed tissue extract (this extract) has an excellent L-PGDS production promoting effect, but there are a wide variety of this extract or preparations containing it. Actions and effects are known. For example, the effects of this extract on the brain include therapeutic effects on ischemic diseases such as cerebral infarction (Japanese Patent Laid-Open No. 2000-16942), and effects on promoting the production of neurotrophic factors such as BDNF (International Publication WO 2011/162317). However, it has not been known so far that this extract has an effect of promoting the production of L-PGDS.
本発明は、脳保護作用、睡眠作用等を有するL-PGDSの産生を促進する物質、及びL-PGDS産生促進作用を指標とする脳保護作用、睡眠作用等を有する薬剤のスクリーニング系等を提供するものである。また、該物質を有効成分として含有し、脳梗塞等の脳血管障害、アルツハイマー病等の認知症、不眠症等のL-PGDSが関与する疾患の予防・治療又は再発予防において、有効で且つ安全性が高い医薬を提供するものである。更には、本発明に係るL-PGDS産生促進剤は、脳血管障害時等における脳の虚血障害、神経細胞傷害等を抑制、緩和する作用を有するものである。なお、本発明において用いられる「治療」には、「軽減」、「改善」、「進行抑制」等の意味が含まれる。また、睡眠作用を有する薬剤を本発明では「睡眠薬」と表記することがあるが、「眠剤」、「睡眠改善薬」、「睡眠導入剤」、「催眠薬」と称するものも本発明に包含され得る。 The present invention provides a substance that promotes the production of L-PGDS that has brain-protective effects, hypnotic effects, etc., and a screening system for drugs that have brain-protective effects, hypnotic effects, etc. using the L-PGDS production promoting effect as an indicator. It is something to do. In addition, it contains this substance as an active ingredient and is effective and safe in the prevention, treatment, or recurrence prevention of diseases related to L-PGDS, such as cerebrovascular disorders such as cerebral infarction, dementia such as Alzheimer's disease, and insomnia. The aim is to provide highly effective medicines. Furthermore, the L-PGDS production promoter according to the present invention has the effect of suppressing and alleviating cerebral ischemic damage, nerve cell injury, etc. during cerebrovascular disorders. Note that the term "treatment" used in the present invention includes meanings such as "alleviation," "improvement," and "suppression of progression." In addition, although drugs having a sleeping effect may be referred to as "sleeping drugs" in the present invention, drugs referred to as "sleeping drugs," "sleep-improving drugs," "sleep-inducing drugs," and "hypnotics" are also included in the present invention. can be included.
本発明者らは、マウス脳梗塞モデル（中大脳動脈永久閉塞モデル）を用いた検討から、血流の遮断によって成熟神経細胞が死滅しつつある梗塞領域内において、神経細胞、アストロサイト、オリゴデンドロサイト等の神経系細胞を含めこれら以外の様々な細胞にも分化し得る幹細胞を見出し、虚血誘導性多能性幹細胞（ischemia-induced multipotent stem cells, iSCs）と命名した。iSCに強発現する神経幹細胞マーカーnestinは、組織化学的には脳軟膜から大脳皮質に至る血管周囲に分布すること、また、iSCはPDGFRβ（血小板由来増殖因子受容体β）やNG2（neuron-glial antigen 2）といった血管周皮細胞（ペリサイト）マーカーを発現すること等から、iSCの由来は血管周囲に分布するペリサイトであると考えられている。ペリサイトは神経細胞やアストロサイト、血管内皮細胞と共に、脳の機能的器質的な基本単位である神経血管単位（neurovascular unit, NVU）を構成し、血液脳関門の形成・維持や神経機能の調節、脳内排出系（glymphatic system）への関与等の重要な役割を担っていると考えられている。iSCは、虚血等の脳傷害時にペリサイトがリプログラミングされることにより生じ、血管内皮細胞等が形成する微小環境によって神経細胞に分化することが示されている。これらのことから、iSCは脳卒中後の血流再建後の神経修復において主要な役割を担う幹細胞であると考えられている。 Based on studies using a mouse cerebral infarction model (middle cerebral artery permanent occlusion model), the present inventors found that neurons, astrocytes, and oligodendrocytes are found in the infarcted area where mature neurons are dying due to blood flow interruption. We discovered stem cells that can differentiate into various other types of cells, including nervous system cells such as cytoplasmic cells, and named them ischemia-induced multipotent stem cells (iSCs). Histochemically, the neural stem cell marker nestin, which is strongly expressed in iSCs, is distributed around blood vessels from the pia mater to the cerebral cortex. It is thought that iSCs originate from pericytes distributed around blood vessels, as they express vascular pericyte markers such as vascular pericyte (pericyte antigen 2). Pericytes, together with neurons, astrocytes, and vascular endothelial cells, constitute the neurovascular unit (NVU), which is the basic functional and organic unit of the brain, and are responsible for the formation and maintenance of the blood-brain barrier and the regulation of nerve function. It is thought to play an important role, such as involvement in the brain's glymphatic system. iSCs are generated by reprogramming of pericytes during brain injury such as ischemia, and have been shown to differentiate into neurons depending on the microenvironment formed by vascular endothelial cells. Based on these findings, iSCs are thought to be stem cells that play a major role in nerve repair after blood flow reconstruction after stroke.
本発明者らは、iSCを用いた研究の一環として、ワクシニアウイルス接種家兎炎症皮膚抽出物（本抽出物）のiSCに対する作用の検討を行った。そして、iSCに本抽出物を添加して培養した後、約2万8千遺伝子のDNAチップによる網羅的遺伝子発現解析を行った結果、本抽出物がL-PGDSをコードする遺伝子PTGDSを選択的に発現促進する作用を有することを見出した。また、タンパク質レベルにおいても、本抽出物はL-PGDSの産生を促進することを確認した。さらに、免疫組織化学的手法により検討した結果、虚血脳に発現するL-PGDSはペリサイトマーカーと共分布することが示された。これらのことから、L-PGDSはペリサイト又はペリサイトから脱分化したiSCに由来するものであると考えられた。 As part of our research using iSCs, the present inventors investigated the effect of a vaccinia virus-inoculated rabbit inflamed skin extract (this extract) on iSCs. After adding this extract to iSCs and culturing them, we performed comprehensive gene expression analysis using a DNA chip of approximately 28,000 genes. As a result, this extract selectively suppressed the gene PTGDS encoding L-PGDS. was found to have the effect of promoting the expression of Furthermore, it was confirmed that this extract also promoted the production of L-PGDS at the protein level. Furthermore, immunohistochemical studies showed that L-PGDS expressed in the ischemic brain was co-distributed with pericyte markers. From these results, it was considered that L-PGDS was derived from pericytes or iSCs dedifferentiated from pericytes.
上述した通り、L-PGDSはPGD2を合成する酵素タンパク質として働くと共に、主として脳内に発現し、様々な疎水性低分子の結合・輸送及びスカベンジャーとしての役割を担って、脳内環境調整・脳保護機能や睡眠調節機能等の様々な機能を有するタンパク質であると考えられている。こうしたことから、L-PGDS産生促進剤は、脳梗塞等の脳血管障害、アルツハイマー病等の認知症、不眠症等のL-PGDSが関与する疾患の予防・治療又は再発予防薬として有用であると考えられる。本抽出物は優れたL-PGDS産生促進作用を有することが確認され、アルツハイマー型認知症モデルマウスの脳内のL-PGDS量を増加させると共に、Aβ量を減少させて、引いては認知機能を改善した。そのため、本抽出物あるいは本抽出物中に含有されるL-PGDS産生促進作用を有する物質、又は本抽出物を含有する製剤は、L-PGDS産生促進剤として大変有用である。 As mentioned above, L-PGDS acts as an enzyme protein that synthesizes PGD2, and is mainly expressed in the brain, and plays the role of binding and transporting various hydrophobic small molecules and as a scavenger, and plays a role in regulating the brain environment and controlling the brain environment. It is thought to be a protein that has various functions such as protective functions and sleep regulating functions. For these reasons, L-PGDS production promoters are useful as prevention/treatment or recurrence prevention agents for diseases related to L-PGDS, such as cerebrovascular disorders such as cerebral infarction, dementia such as Alzheimer's disease, and insomnia. it is conceivable that. This extract has been confirmed to have an excellent L-PGDS production promoting effect, increasing the amount of L-PGDS in the brain of Alzheimer's disease model mice and decreasing the amount of Aβ, which in turn improves cognitive function. improved. Therefore, this extract, a substance contained in this extract that has an L-PGDS production promoting effect, or a preparation containing this extract is very useful as an L-PGDS production promoter.
また、本発明者らは、iSCにおけるL-PGDS産生促進作用を指標として、L-PGDSが関与する疾患の治療に有用な薬剤のスクリーニング方法として有用であることを見出した。当該スクリーニング方法は、特に脳保護作用や睡眠作用を有する薬剤の開発に寄与することが考えられる。また、本発明は、iSCにおけるL-PGDS産生促進作用を指標として、本抽出物又は本抽出物を含有する製剤を試験することにより、本抽出物又は本抽出物を含有する製剤の作用・効果を判定又は評価でき、引いては本抽出物又は本抽出物を含有する製剤の薬効を担保する方法を提供する。本発明者らは、以上の知見に基づき、本発明を完成させた。 Furthermore, the present inventors have found that the method of promoting L-PGDS production in iSCs is useful as a screening method for drugs useful in treating diseases involving L-PGDS. It is thought that the screening method will particularly contribute to the development of drugs that have brain-protective and hypnotic effects. In addition, the present invention examines the effects and effects of the present extract or preparations containing the present extract by testing the present extract or preparations containing the present extract using the L-PGDS production promoting effect in iSCs as an indicator. The present invention provides a method that enables determination or evaluation of the present extract and also ensures the medicinal efficacy of the present extract or a preparation containing the present extract. The present inventors completed the present invention based on the above findings.
すなわち、本願発明には、例えば下記の態様が包含される。
（１）リポカリン型プロスタグランジンD2合成酵素産生促進剤。
（２）ワクシニアウイルス接種炎症組織抽出物が含有する、上記（１）に記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（３）ワクシニアウイルス接種炎症組織抽出物を含有する、上記（１）に記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（４）リポカリン型プロスタグランジンD2合成酵素がペリサイト又はペリサイトから脱分化した虚血誘導性多能性幹細胞において産生されるものである、上記（１）～（３）のいずれかに記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（５）脳保護薬である、上記（１）～（４）のいずれかに記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（６）脳保護薬の作用が脳内排出系に対する亢進作用によるものである、上記（５）に記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（７）脳保護薬が脳梗塞の予防・治療又は再発予防薬である、上記（５）又は（６）に記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（８）脳保護薬が認知症の予防・治療薬である、上記（５）又は（６）に記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（９）認知症がアルツハイマー型認知症である、上記（８）に記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（１０）アミロイドβ沈着の阻害作用を有する、上記（９）に記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（１１）睡眠薬である、上記（１）～（４）のいずれかに記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（１２）炎症組織がウサギの炎症皮膚組織である、上記（２）～（１１）のいずれかに記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（１３）注射剤である、上記（１）～（１２）のいずれかに記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
（１４）経口剤である、上記（１）～（１２）のいずれかに記載のリポカリン型プロスタグランジンD2合成酵素産生促進剤。
That is, the present invention includes, for example, the following aspects.
(1) Lipocalin-type prostaglandin D2 synthase production promoter.
(2) The lipocalin-type prostaglandin D2 synthase production promoter according to (1) above, which is contained in the vaccinia virus-inoculated inflamed tissue extract.
(3) The lipocalin-type prostaglandin D2 synthase production promoter according to (1) above, which contains an extract of inflamed tissue inoculated with vaccinia virus.
(4) The lipocalin-type prostaglandin D2 synthase is produced in pericytes or ischemia-induced pluripotent stem cells dedifferentiated from pericytes, according to any one of (1) to (3) above. lipocalin-type prostaglandin D2 synthase production promoter.
(5) The lipocalin-type prostaglandin D2 synthase production promoter according to any one of (1) to (4) above, which is a brain protective drug.
(6) The lipocalin-type prostaglandin D2 synthase production promoter according to (5) above, wherein the action of the brain protective drug is due to an enhancing action on the brain excretion system.
(7) The lipocalin-type prostaglandin D2 synthase production promoter according to (5) or (6) above, wherein the brain protective drug is a drug for preventing/treating cerebral infarction or preventing recurrence.
(8) The lipocalin-type prostaglandin D2 synthase production promoter according to (5) or (6) above, wherein the brain protective drug is a preventive/therapeutic drug for dementia.
(9) The lipocalin-type prostaglandin D2 synthase production promoter according to (8) above, wherein the dementia is Alzheimer's type dementia.
(10) The lipocalin-type prostaglandin D2 synthase production promoter according to (9) above, which has an inhibitory effect on amyloid β deposition.
(11) The lipocalin-type prostaglandin D2 synthase production promoter according to any one of (1) to (4) above, which is a sleeping pill.
(12) The lipocalin-type prostaglandin D2 synthase production promoter according to any one of (2) to (11) above, wherein the inflamed tissue is rabbit inflamed skin tissue.
(13) The lipocalin-type prostaglandin D2 synthase production promoter according to any one of (1) to (12) above, which is an injection.
(14) The lipocalin-type prostaglandin D2 synthase production promoter according to any one of (1) to (12) above, which is an oral preparation.
（１５）リポカリン型プロスタグランジンD2合成酵素の発現促進作用を指標とする、脳保護作用又は睡眠作用を有する物質のスクリーニング方法。
（１６）ペリサイト又はペリサイトから脱分化した虚血誘導性多能性幹細胞におけるリポカリン型プロスタグランジンD2合成酵素の発現促進作用を指標とする、上記（１５）に記載のスクリーニング方法。
（１７）脳保護作用を有する物質が、脳梗塞の予防・治療若しくは再発予防薬又は認知症の予防・治療薬である、上記（１５）又は（１６）に記載のスクリーニング方法。
（１８）認知症がアルツハイマー型認知症である上記（１７）に記載のスクリーニング方法。
（１９）睡眠作用を有する物質が、睡眠薬である上記（１５）又は（１６）に記載のスクリーニング方法。
（２０）上記（１５）～（１９）のいずれかに記載のスクリーニング方法によって得られた脳保護作用又は睡眠作用を有する物質。
（２１）脳保護作用がアミロイドβの排出作用によるものである、上記（２０）に記載の物質。
(15) A method for screening for a substance having a brain protective effect or a hypnotic effect, using as an indicator the expression promoting effect of lipocalin-type prostaglandin D2 synthase.
(16) The screening method according to (15) above, which uses as an indicator the expression promoting effect of lipocalin-type prostaglandin D2 synthase in pericytes or ischemia-induced pluripotent stem cells dedifferentiated from pericytes.
(17) The screening method according to (15) or (16) above, wherein the substance having a brain protective effect is a preventive/therapeutic agent for cerebral infarction or recurrence, or a preventive/therapeutic agent for dementia.
(18) The screening method according to (17) above, wherein the dementia is Alzheimer's type dementia.
(19) The screening method according to (15) or (16) above, wherein the substance having a hypnotic effect is a sleeping pill.
(20) A substance having a brain protective effect or a sleep effect obtained by the screening method according to any one of (15) to (19) above.
(21) The substance according to (20) above, wherein the brain protective effect is due to the action of excreting amyloid β.
（２２）リポカリン型プロスタグランジンD2合成酵素の発現促進作用を指標とする、ワクシニアウイルス接種炎症組織抽出物又はこれを含有する製剤の判定又は評価方法。
（２３）ペリサイト又はペリサイトから脱分化した虚血誘導性多能性幹細胞におけるリポカリン型プロスタグランジンD2合成酵素の発現促進作用を指標とする、上記（２２）に記載の判定又は評価方法。
（２４）炎症組織がウサギの炎症皮膚組織である上記（２２）又は（２３）に記載の判定又は評価方法。
（２５）上記（２２）～（２４）のいずれかに記載の判定又は評価を行うことによってワクシニアウイルス接種炎症組織抽出物又はこれを含有する製剤の品質規格を担保する方法。
（２６）上記（２２）～（２４）のいずれかに記載の判定又は評価を行うことによって品質規格が担保されたワクシニアウイルス接種炎症組織抽出物又はこれを含有する製剤。
(22) A method for determining or evaluating a vaccinia virus-inoculated inflamed tissue extract or a preparation containing the same, using the expression-promoting effect of lipocalin-type prostaglandin D2 synthase as an index.
(23) The determination or evaluation method according to (22) above, wherein the expression promoting effect of lipocalin-type prostaglandin D2 synthase in pericytes or ischemia-induced pluripotent stem cells dedifferentiated from pericytes is used as an indicator.
(24) The determination or evaluation method according to (22) or (23) above, wherein the inflamed tissue is rabbit inflamed skin tissue.
(25) A method for ensuring the quality standards of a vaccinia virus-inoculated inflamed tissue extract or a preparation containing the same by performing the determination or evaluation described in any of (22) to (24) above.
(26) A vaccinia virus-inoculated inflamed tissue extract or a preparation containing the same, whose quality standards are ensured by performing the determination or evaluation described in any of (22) to (24) above.
（２７）ワクシニアウイルス接種炎症組織抽出物の有効量を当該治療を必要とする患者に投与することからなるリポカリン型プロスタグランジンD2合成酵素の産生促進方法。
（２８）リポカリン型プロスタグランジンD2合成酵素がペリサイト又はペリサイトから脱分化した虚血誘導性多能性幹細胞において産生されるものである、上記（２７）に記載のリポカリン型プロスタグランジンD2合成酵素の産生促進方法。
（２９）リポカリン型プロスタグランジンD2合成酵素の産生促進が脳保護又は睡眠導入・改善のために作用する、上記（２７）又は（２８）に記載のリポカリン型プロスタグランジンD2合成酵素の産生促進方法。
（３０）脳保護が脳梗塞の予防・治療又は再発予防である、上記（２９）に記載のリポカリン型プロスタグランジンD2合成酵素の産生促進方法。
（３１）脳保護が認知症の予防・治療である、上記（２９）に記載のリポカリン型プロスタグランジンD2合成酵素の産生促進方法。
（３２）認知症がアルツハイマー型認知症である、上記（３１）に記載のリポカリン型プロスタグランジンD2合成酵素の産生促進方法。
(27) A method for promoting the production of lipocalin-type prostaglandin D2 synthase, which comprises administering an effective amount of an extract of an inflamed tissue inoculated with vaccinia virus to a patient in need of the treatment.
(28) The lipocalin-type prostaglandin D2 according to (27) above, wherein the lipocalin-type prostaglandin D2 synthase is produced in pericytes or ischemia-induced pluripotent stem cells dedifferentiated from pericytes. Method for promoting production of synthetic enzymes.
(29) Promoting the production of lipocalin-type prostaglandin D2 synthase according to (27) or (28) above, wherein the promotion of production of lipocalin-type prostaglandin D2 synthase acts to protect the brain or induce/improve sleep. Method.
(30) The method for promoting production of lipocalin-type prostaglandin D2 synthase according to (29) above, wherein the brain protection is prevention/treatment of cerebral infarction or prevention of recurrence.
(31) The method for promoting production of lipocalin-type prostaglandin D2 synthase according to (29) above, wherein brain protection is prevention/treatment of dementia.
(32) The method for promoting production of lipocalin-type prostaglandin D2 synthase according to (31) above, wherein the dementia is Alzheimer's type dementia.
（３３）リポカリン型プロスタグランジンD2合成酵素の産生促進に用いるためのワクシニアウイルス接種炎症組織抽出物。
（３４）リポカリン型プロスタグランジンD2合成酵素がペリサイト又はペリサイトから脱分化した虚血誘導性多能性幹細胞において産生されるものである、上記（３３）に記載のワクシニアウイルス接種炎症組織抽出物。
（３５）リポカリン型プロスタグランジンD2合成酵素の産生促進が脳保護又は睡眠導入・改善に用いるためである、上記（３３）又は（３４）に記載のワクシニアウイルス接種炎症組織抽出物。
（３６）脳保護が脳梗塞の予防・治療又は再発予防である、上記（３５）に記載のワクシニアウイルス接種炎症組織抽出物。
（３７）脳保護が認知症の予防・治療である、上記（３５）に記載のワクシニアウイルス接種炎症組織抽出物。
（３８）認知症がアルツハイマー型認知症である、上記（３７）に記載のワクシニアウイルス接種炎症組織抽出物。
(33) An extract of inflamed tissue inoculated with vaccinia virus for use in promoting the production of lipocalin-type prostaglandin D2 synthase.
(34) The vaccinia virus-inoculated inflamed tissue extraction according to (33) above, wherein lipocalin-type prostaglandin D2 synthase is produced in pericytes or ischemia-induced pluripotent stem cells dedifferentiated from pericytes. thing.
(35) The vaccinia virus-inoculated inflamed tissue extract according to (33) or (34) above, which promotes the production of lipocalin-type prostaglandin D2 synthase and is used for brain protection or sleep induction/improvement.
(36) The vaccinia virus-inoculated inflamed tissue extract according to (35) above, wherein the brain protection is prevention/treatment of cerebral infarction or prevention of recurrence.
(37) The vaccinia virus-inoculated inflamed tissue extract according to (35) above, whose brain protection is prevention/treatment of dementia.
(38) The vaccinia virus-inoculated inflamed tissue extract according to (37) above, wherein the dementia is Alzheimer's type dementia.
（３９）リポカリン型プロスタグランジンD2合成酵素の産生を促進する医薬の製造におけるワクシニアウイルス接種炎症組織抽出物の使用。
（４０）リポカリン型プロスタグランジンD2合成酵素がペリサイト又はペリサイトから脱分化した虚血誘導性多能性幹細胞において産生されるものである、上記（３９）に記載の使用。
（４１）リポカリン型プロスタグランジンD2合成酵素の産生促進する医薬が脳保護薬又は睡眠薬である、上記（３９）又は（４０）に記載の使用。
（４２）脳保護薬が脳梗塞の予防・治療又は再発予防薬である、上記（４１）に記載の使用。
（４３）脳保護薬が認知症の予防・治療薬である、上記（４１）に記載の使用。
（４４）認知症がアルツハイマー型認知症である、上記（４３）に記載の使用。
(39) Use of a vaccinia virus-inoculated inflamed tissue extract in the manufacture of a medicament that promotes the production of lipocalin-type prostaglandin D2 synthase.
(40) The use according to (39) above, wherein the lipocalin-type prostaglandin D2 synthase is produced in pericytes or ischemia-induced pluripotent stem cells dedifferentiated from pericytes.
(41) The use according to (39) or (40) above, wherein the drug that promotes production of lipocalin-type prostaglandin D2 synthase is a brain protective drug or a sleeping pill.
(42) The use according to (41) above, wherein the brain protective drug is a drug for preventing/treating cerebral infarction or preventing recurrence.
(43) The use according to (41) above, wherein the brain protective drug is a preventive/therapeutic drug for dementia.
(44) The use according to (43) above, wherein the dementia is Alzheimer's type dementia.
本発明L-PGDS産生促進剤は、ペリサイト又はiSCの発現するL-PGDSのリポカリンとしての機能を亢進することにより、脳内の様々な疎水性分子を排出するトランスポーターとして働いて、脳虚血時の傷害や認知症の原因とされる物質による弊害から脳を保護する作用を発揮することが強く期待される。また、本発明L-PGDS産生促進剤は、細胞内で合成したPGD2を脳脊髄液中に分泌し、脳内のPGD2レセプターへと輸送することにより睡眠調節作用等を発揮することが期待される。特に、本抽出物は、マウスの虚血脳において、L-PGDSの産生を促進することが明らかにされ、さらに、本抽出物をアルツハイマー型認知症モデルマウスに投与することにより、脳内のL-PGDS量が上昇すると共に、Aβ量が減少し、認知機能が改善することが確認された。このように、本抽出物がL-PGDSの産生を促進させて優れた薬理作用を有することは動物実験でも認められている。また、本抽出物を含有する製剤は、副作用等の問題点の少ない安全性の高い薬剤として長年使用されているものである。そのため、本発明は極めて有用性の高いものである。 The L-PGDS production promoter of the present invention enhances the lipocalin function of L-PGDS expressed by pericytes or iSCs, thereby acting as a transporter to excrete various hydrophobic molecules in the brain, resulting in brain damage. It is strongly expected that it will protect the brain from the harmful effects of blood damage and substances that are thought to cause dementia. Furthermore, the L-PGDS production promoter of the present invention is expected to exert sleep regulating effects by secreting PGD2 synthesized within cells into the cerebrospinal fluid and transporting it to PGD2 receptors in the brain. . In particular, this extract was shown to promote the production of L-PGDS in the ischemic brain of mice, and furthermore, by administering this extract to Alzheimer's disease model mice, L-PGDS in the brain was stimulated. -It was confirmed that as the PGDS level increased, the Aβ level decreased and cognitive function improved. Thus, it has been confirmed in animal experiments that this extract promotes the production of L-PGDS and has excellent pharmacological effects. Furthermore, preparations containing this extract have been used for many years as highly safe drugs with few problems such as side effects. Therefore, the present invention is extremely useful.
また、本抽出物は非常に膨大な数の成分からなる多成分系の物質であることから、その作用を単一あるいは複数の含有成分の含量等で判定又は評価し、薬効等を担保することは極めて困難である。その点、本発明のL-PGDSの発現促進作用を指標とする本抽出物又は本抽出物を含有する製剤の判定又は評価方法によれば、簡便に本抽出物又は本抽出物を含有する製剤の作用を判定又は評価でき、引いては本抽出物又はこれを含有する製剤の薬効を担保することに利用できる。この点でも本発明は有用性の高いものである。なお、本願における「判定又は評価」には、試験、検査等により対象物を調べて効果、作用、適否等を見定める概念のすべてが包含されるものである。 In addition, since this extract is a multicomponent substance consisting of an extremely large number of components, it is necessary to determine or evaluate its action based on the content of a single or multiple components to ensure its medicinal efficacy. is extremely difficult. In this regard, according to the method for determining or evaluating the present extract or a preparation containing the present extract using the L-PGDS expression promoting effect of the present invention as an indicator, the present extract or a preparation containing the present extract can be easily obtained. It can be used to determine or evaluate the effect of the present extract or to ensure the medicinal efficacy of the extract or a preparation containing the same. The present invention is also highly useful in this respect. Note that "judgment or evaluation" in this application includes all concepts of examining an object through tests, inspections, etc. to determine effects, actions, suitability, etc.
本抽出物は、ワクシニアウイルスを接種して発痘した動物の炎症組織から抽出分離した非蛋白性の活性物質を含有する抽出物である。本抽出物は、抽出された状態では液体であるが、乾燥することにより固体にすることもできる。本製剤は、医薬品として非常に有用なものである。本製剤として出願人が日本において製造し販売している具体的な商品に「ワクシニアウイルス接種家兎炎症皮膚抽出液含有製剤」（商品名：ノイロトロピン／NEUROTROPIN〔登録商標〕）（以下「ノイロトロピン」という。）がある。ノイロトロピンには、注射剤と錠剤があり、いずれも医療用医薬品（ethical drug）である。 This extract is an extract containing non-protein active substances extracted and separated from the inflamed tissue of an animal that has been inoculated with vaccinia virus and has developed pox. The extract is liquid in the extracted state, but can also be made into a solid by drying. This preparation is very useful as a pharmaceutical. The specific product that the applicant manufactures and sells in Japan as this preparation is "Preparation containing extract of inflamed skin of rabbits inoculated with vaccinia virus" (trade name: NEUROTROPIN [registered trademark]) (hereinafter referred to as "Neurotropin"). ). Neurotropin comes in injections and tablets, both of which are ethical drugs.
ノイロトロピンの注射剤の適応症は、「腰痛症、頸肩腕症候群、症候性神経痛、皮膚疾患（湿疹、皮膚炎、蕁麻疹）に伴う掻痒、アレルギー性鼻炎、スモン（SMON）後遺症状の冷感・異常知覚・痛み」である。ノイロトロピンの錠剤の適応症は、「帯状疱疹後神経痛、腰痛症、頸肩腕症候群、肩関節周囲炎、変形性関節症」である。本製剤は、出願人が創製し、医薬品として開発したものであり、その有効性と安全性における優れた特長が評価され、長年にわたり販売されて、日本の医薬品市場で確固たる地位を確立しているものである。 The indications for neurotropin injections are low back pain, cervico-shoulder-brachial syndrome, symptomatic neuralgia, pruritus associated with skin diseases (eczema, dermatitis, urticaria), allergic rhinitis, cold sensation and after-effects of SMON. ``Abnormal perception/pain.'' Neurotropin tablets are indicated for ``postherpetic neuralgia, low back pain, cervico-shoulder-brachial syndrome, glenohumeral periarthritis, and osteoarthritis.'' This preparation was created by the applicant and developed as a pharmaceutical product.It has been highly evaluated for its excellent features in terms of effectiveness and safety, and has been sold for many years, establishing a firm position in the Japanese pharmaceutical market. It is something.
本発明におけるワクシニアウイルス接種炎症組織抽出物はワクシニアウイルスを接種して発痘した炎症組織を破砕し、抽出溶媒を加えて組織片を除去した後、除蛋白処理を行い、これを吸着剤に吸着させ、次いで有効成分を溶出することによって得ることができる。即ち、例えば、以下のような工程である。
（A）ワクシニアウイルスを接種し発痘させたウサギ、マウス等の皮膚組織等を採取し、発痘組織を破砕し、水、フェノール水、生理食塩液またはフェノール加グリセリン水等の抽出溶媒を加えた後、濾過または遠心分離することによって抽出液（濾液または上清）を得る。
（B）前記抽出液を酸性のpHに調整して加熱し、除蛋白処理する。次いで除蛋白した溶液をアルカリ性に調整して加熱した後に濾過または遠心分離する。
（C）得られた濾液または上清を酸性とし活性炭、カオリン等の吸着剤に吸着させる。
（D）前記吸着剤に水等の抽出溶媒を加え、アルカリ性のpHに調整し、吸着成分を溶出することによってワクシニアウイルス接種炎症組織抽出物を得ることができる。その後、所望に応じて、適宜溶出液を減圧下に蒸発乾固または凍結乾燥することによって乾固物とすることもできる。
The extract of inflamed tissue inoculated with vaccinia virus in the present invention is obtained by crushing the inflamed tissue that has been inoculated with vaccinia virus, removing tissue fragments by adding an extraction solvent, and then deproteinizing the tissue, which is then adsorbed onto an adsorbent. It can be obtained by allowing the active ingredient to dissolve and then eluting the active ingredient. That is, for example, the steps are as follows.
(A) Collect skin tissues from rabbits, mice, etc. that have been inoculated with vaccinia virus and caused pox, crush the pox tissue, and add an extraction solvent such as water, phenol water, physiological saline, or phenol-added glycerol water. After that, an extract (filtrate or supernatant) is obtained by filtration or centrifugation.
(B) The extract is adjusted to an acidic pH and heated to remove protein. Next, the protein-removed solution is adjusted to alkalinity, heated, and then filtered or centrifuged.
(C) The obtained filtrate or supernatant is acidified and adsorbed onto an adsorbent such as activated carbon or kaolin.
(D) A vaccinia virus-inoculated inflamed tissue extract can be obtained by adding an extraction solvent such as water to the adsorbent, adjusting the pH to alkaline, and eluting the adsorbed components. Thereafter, if desired, the eluate can be evaporated to dryness or freeze-dried under reduced pressure to obtain a dry solid.
ワクシニアウイルスを接種し炎症組織を得るための動物としては、ウサギ、ウシ、ウマ、ヒツジ、ヤギ、サル、ラット、マウスなどワクシニアウイルスが感染する種々の動物を用いることができ、炎症組織としてはウサギの炎症皮膚組織が好ましい。ウサギはウサギ目に属するものであればいかなるものでもよい。例としては、アナウサギ、カイウサギ（アナウサギを家畜化したもの）、ノウサギ（ニホンノウサギ）、ナキウサギ、ユキウサギ等がある。これらのうち、カイウサギが使用するには好適である。日本では過去から飼育され家畜又は実験用動物として繁用されている家兎（イエウサギ）と呼ばれるものがあるが、これもカイウサギの別称である。カイウサギには、多数の品種（ブリード）が存在するが、日本白色種やニュージーランド白色種（ニュージーランドホワイト）といった品種が好適に用いられ得る。 Various animals that can be infected with vaccinia virus, such as rabbits, cows, horses, sheep, goats, monkeys, rats, and mice, can be used to inoculate vaccinia virus and obtain inflamed tissue. of inflamed skin tissue is preferred. The rabbit may be any species as long as it belongs to the order Lagomorpha. Examples include rabbit rabbits, rabbit rabbits (domesticated rabbits), hares (Japanese hares), pika rabbits, and snow rabbits. Among these, it is suitable for use by rabbits. In Japan, there is a species called the domestic rabbit, which has been bred for a long time and is often used as livestock or experimental animals, and this is also another name for the rabbit. There are many breeds of rabbits, and breeds such as the Japanese white breed and the New Zealand white breed (New Zealand white) can be preferably used.
ワクシニアウイルス（vaccinia virus）は、いかなる株のものであってもよい。例としては、リスター（Lister）株、大連（Dairen）株、池田（Ikeda）株、ＥＭ－６３株、ニューヨーク市公衆衛生局（New York City Board of Health）株等が挙げられる。 The vaccinia virus may be of any strain. Examples include Lister strain, Dairen strain, Ikeda strain, EM-63 strain, New York City Board of Health strain, and the like.
上記した本抽出物の基本的な抽出工程（A）～（D）は、より詳しくは、例えば、以下のようなものとして実施できる。
工程（A）について
ウサギの皮膚にワクシニアウイルスを皮内接種して発痘させた炎症皮膚組織を採取する。採取した皮膚組織はフェノール溶液等で洗浄、消毒を行なう。この炎症皮膚組織を破砕し、その1乃至5倍量の抽出溶媒を加える。ここで、破砕とは、ミンチ機等を使用してミンチ状に細かく砕くことを意味する。また、抽出溶媒としては、蒸留水、生理食塩水、弱酸性乃至弱塩基性の緩衝液などを用いることができ、フェノール等の殺菌・防腐剤、グリセリン等の安定化剤、塩化ナトリウム、塩化カリウム、塩化マグネシウム等の塩類などを適宜添加してもよい。この時、凍結融解、超音波、細胞膜溶解酵素又は界面活性剤等の処理により細胞組織を破壊して抽出を容易にすることもできる。得られた懸濁液を、5日乃至12日間放置する。その間、適宜攪拌しながら又は攪拌せずに、30乃至45℃に加温してもよい。得られた液を固液分離（濾過又は遠心分離等）によって組織片を除去して粗抽出液（濾液又は上清）を得る。
More specifically, the basic extraction steps (A) to (D) of the present extract described above can be carried out, for example, as follows.
Regarding step (A): Vaccinia virus is intradermally inoculated into the skin of a rabbit to cause pox and inflamed skin tissue is collected. The collected skin tissue is washed and disinfected with a phenol solution. The inflamed skin tissue is crushed and 1 to 5 times the volume of extraction solvent is added. Here, crushing means finely crushing into minced pieces using a mincing machine or the like. In addition, distilled water, physiological saline, weakly acidic or weakly basic buffers, etc. can be used as extraction solvents, disinfectants and preservatives such as phenol, stabilizers such as glycerin, sodium chloride, potassium chloride, etc. , salts such as magnesium chloride, etc. may be added as appropriate. At this time, the cell tissue can be destroyed to facilitate extraction by freezing and thawing, ultrasound, treatment with a cell membrane lytic enzyme, a surfactant, or the like. The resulting suspension is left for 5 to 12 days. During this time, the mixture may be heated to 30 to 45° C. with or without stirring as appropriate. Tissue pieces are removed from the resulting liquid by solid-liquid separation (filtration, centrifugation, etc.) to obtain a crude extract (filtrate or supernatant).
工程（B）について
工程（A）で得られた粗抽出液について除蛋白処理を行う。除蛋白は、通常行われている公知の方法により実施でき、加熱処理、蛋白質変性剤（例えば、酸、塩基、尿素、グアニジン、アセトン等の有機溶媒など）による処理、等電点沈澱、塩析等の方法を適用することができる。次いで、不溶物を除去する通常の方法、例えば、濾紙（セルロース、ニトロセルロース等）、グラスフィルター、セライト、ザイツ濾過板等を用いた濾過、限外濾過、遠心分離などにより析出してきた不溶蛋白質を除去した濾液又は上清を得る。
Regarding step (B): The crude extract obtained in step (A) is subjected to protein removal treatment. Protein removal can be carried out by commonly known methods, including heat treatment, treatment with protein denaturants (e.g., acids, bases, urea, guanidine, organic solvents such as acetone, etc.), isoelectric precipitation, and salting out. Methods such as the following can be applied. Next, the precipitated insoluble protein is removed by a conventional method for removing insoluble matter, such as filtration using filter paper (cellulose, nitrocellulose, etc.), glass filter, Celite, Seitz filter plate, etc., ultrafiltration, centrifugation, etc. Obtain the removed filtrate or supernatant.
工程（C）について
工程（B）で得られた濾液又は上清を、酸性、好ましくはpH3.5乃至5.5に調整し、吸着剤への吸着操作を行う。使用可能な吸着剤としては、活性炭、カオリン等を挙げることができ、抽出液中に吸着剤を添加し撹拌するか、抽出液を吸着剤充填カラムに通過させて、該吸着剤に有効成分を吸着させることができる。抽出液中に吸着剤を添加した場合には、濾過や遠心分離等によって溶液を除去して、活性成分を吸着させた吸着剤を得ることができる。
Regarding Step (C) The filtrate or supernatant obtained in Step (B) is acidified, preferably adjusted to pH 3.5 to 5.5, and adsorbed onto an adsorbent. Adsorbents that can be used include activated carbon, kaolin, etc. The adsorbent is added to the extract and stirred, or the extract is passed through an adsorbent-filled column to absorb the active ingredients into the adsorbent. It can be adsorbed. When an adsorbent is added to the extract, the solution can be removed by filtration, centrifugation, etc. to obtain an adsorbent on which active ingredients have been adsorbed.
工程（D）について
工程（C）で得られた吸着剤から活性成分を溶出（脱離）させるには、当該吸着剤に溶出溶媒を加え、塩基性、好ましくはpH9乃至12に調整し、室温又は適宜加熱して或いは撹拌して溶出し、濾過や遠心分離等の通常の方法で吸着剤を除去する。用いられる溶出溶媒としては、塩基性の溶媒、例えば塩基性のpHに調整した水、メタノール、エタノール、イソプロパノール等又はこれらの適当な混合溶液を用いることができ、好ましくはpH9乃至12に調整した水を使用することができる。溶出溶媒の量は適宜設定することができる。このようにして得られた溶出液を、原薬として用いるために、適宜pHを中性付近に調整するなどして、最終的にワクシニアウイルス接種ウサギ炎症皮膚抽出物（本抽出物）を得ることができる。
Regarding step (D) To elute (desorb) the active ingredient from the adsorbent obtained in step (C), add an elution solvent to the adsorbent, adjust it to basicity, preferably pH 9 to 12, and leave it at room temperature. Alternatively, the adsorbent is eluted by heating or stirring as appropriate, and the adsorbent is removed by a conventional method such as filtration or centrifugation. As the elution solvent used, a basic solvent such as water adjusted to a basic pH, methanol, ethanol, isopropanol, etc., or an appropriate mixed solution thereof can be used, preferably water adjusted to a pH of 9 to 12. can be used. The amount of elution solvent can be set appropriately. In order to use the eluate obtained in this way as a drug substance, the pH is appropriately adjusted to around neutrality to finally obtain a vaccinia virus-inoculated rabbit inflamed skin extract (this extract). Can be done.
本抽出物は、できた時点では液体であるので、適宜濃縮・希釈することによって所望の濃度のものにすることもできる。本抽出物から製剤を製造する場合には、加熱滅菌処理を施すのが好ましい。注射剤にするためには、例えば塩化ナトリウム等を加えて生理食塩液と等張の溶液に調製することができる。また、液体あるいはゲル等の状態で経口投与することも可能であるが、本抽出物に適切な濃縮乾固等の操作を行うことによって、錠剤等の経口用固形製剤を製造することもできる。本抽出物からこのような経口用固形製剤を製造する具体的な方法は、日本特許第3818657号や同第4883798号の明細書に記載されている。こうして得られる注射剤や経口用製剤等が本製剤の例である。 Since this extract is a liquid at the time of preparation, it can be concentrated and diluted as appropriate to obtain a desired concentration. When producing a preparation from this extract, it is preferable to perform heat sterilization treatment. To prepare an injection, for example, sodium chloride or the like can be added to prepare a solution that is isotonic with physiological saline. Although it is possible to orally administer the extract in the form of a liquid or gel, it is also possible to produce solid oral preparations such as tablets by subjecting the extract to appropriate operations such as concentration-drying. A specific method for producing such an oral solid preparation from this extract is described in the specifications of Japanese Patent No. 3818657 and Japanese Patent No. 4883798. Injections and oral preparations obtained in this manner are examples of the present preparation.
患者への投与方法としては、特に限定されず、治療目的に応じて適宜選択することができる。例えば、経口投与の他に皮下、筋肉内、静脈内投与、経皮投与等が挙げられ、投与量はワクシニアウイルス接種炎症組織抽出物の種類によって適宜設定することができる。市販の製剤で認められている投与量は、基本的には内服では1日16NU、注射剤では1日3.6乃至7.2NUを投与するよう医療用医薬品としては示されているが、疾患の種類、重傷度、患者の個人差、投与方法、投与期間等によって適宜増減可能である（NU：ノイロトロピン単位。ノイロトロピン単位とは、疼痛閾値が正常動物より低下した慢性ストレス動物であるSARTストレスマウスを用い、Randall-Selitto変法に準じて試験を行い、鎮痛効力のED50値をもって規定する。1NUはED50値が100 mg／kgであるときのノイロトロピン製剤の鎮痛活性含有成分1mgを示す活性である。）。 The method of administration to patients is not particularly limited and can be appropriately selected depending on the therapeutic purpose. For example, in addition to oral administration, subcutaneous, intramuscular, intravenous, and transdermal administration may be used, and the dosage can be appropriately determined depending on the type of inflamed tissue extract inoculated with vaccinia virus. The approved dosage for commercially available preparations is basically 16 NU per day for oral administration, and 3.6 to 7.2 NU per day for injections as a medical drug, but depending on the type of disease, The dose can be adjusted as appropriate depending on the degree of severity, individual differences in patients, administration method, administration period, etc. (NU: neurotropin unit. Neurotropin unit is a chronically stressed animal with a pain threshold lower than that of a normal animal, using SART stressed mice. The test is conducted according to the modified Randall-Selitto method, and the analgesic efficacy is defined by the ED50 value. 1 NU is the activity representing 1 mg of the analgesic active ingredient of the neurotropin preparation when the ED50 value is 100 mg/kg).
以下に、本抽出物の製造方法の例、及び本抽出物の新規な薬理作用、L-PGDS産生促進作用に関する薬理試験結果を示すが、本発明はこれらの実施例の記載によって何ら制限されるものではない。 Examples of the method for producing the present extract, and pharmacological test results regarding the novel pharmacological action and L-PGDS production promoting action of the present extract are shown below, but the present invention is not limited in any way by the description of these Examples. It's not a thing.
実施例１ 本抽出物の製造
健康な成熟家兎の皮膚にワクシニアウイルスを皮内接種し、発痘した皮膚を切り取り採取した。採取した皮膚はフェノール溶液で洗浄・消毒を行なった後、余分のフェノール溶液を除去し、破砕して、フェノール溶液を加え混合し、3～７日間放置した後、さらに3～4日間攪拌しながら35～40℃に加温した。その後、固液分離して得た抽出液を塩酸でpH4.5～5.2に調整し、90～100℃で30分間、加熱処理した後、濾過して除蛋白した。さらに、濾液を水酸化ナトリウムでpH9.0～9.5に調整し、90～100℃で15分間、加熱処理した後、固液分離した。
Example 1 Production of the present extract Vaccinia virus was intradermally inoculated into the skin of a healthy adult rabbit, and the infected skin was cut and collected. The collected skin was washed and disinfected with a phenol solution, the excess phenol solution was removed, the skin was crushed, the phenol solution was added and mixed, the skin was left for 3 to 7 days, and then the skin was crushed for another 3 to 4 days with stirring. It was heated to 35-40°C. Thereafter, the extract obtained by solid-liquid separation was adjusted to pH 4.5-5.2 with hydrochloric acid, heated at 90-100°C for 30 minutes, and then filtered to remove protein. Furthermore, the filtrate was adjusted to pH 9.0 to 9.5 with sodium hydroxide, heated at 90 to 100°C for 15 minutes, and then separated into solid and liquid.
得られた除蛋白液を塩酸でpH4.0～4.3に調整し、除蛋白液質量の2％量の活性炭を加えて2時間撹拌した後、固液分離した。採取した活性炭に水を加え、水酸化ナトリウムでpH9.5～10とし、60℃で90～100分間撹拌した後、遠心分離して上清を得た。遠心分離で沈澱した活性炭に再び水を加えた後、水酸化ナトリウムでpH10.5～11とし、60℃で90～100分間撹拌した後、遠心分離して上清を得た。両上清を合せて、塩酸で中和し、本抽出物を得た。 The resulting protein removal solution was adjusted to pH 4.0 to 4.3 with hydrochloric acid, activated carbon was added in an amount of 2% of the weight of the protein removal solution, stirred for 2 hours, and then solid-liquid separated. Water was added to the collected activated carbon, the pH was adjusted to 9.5-10 with sodium hydroxide, the mixture was stirred at 60°C for 90-100 minutes, and then centrifuged to obtain a supernatant. After adding water again to the activated carbon precipitated by centrifugation, the pH was adjusted to 10.5-11 with sodium hydroxide, stirred at 60°C for 90-100 minutes, and then centrifuged to obtain a supernatant. Both supernatants were combined and neutralized with hydrochloric acid to obtain the present extract.
実施例２ 薬理試験
次に、上記実施例１で得られた本抽出物を被験物質として用いたL-PGDS産生促進作用に関する薬理試験の方法及び結果を示す。なお、下記の薬理試験において、C.B-17マウスへの脳梗塞の導入や、該脳梗塞巣からのiSCの単離培養については、Nakagomi, T. et al. Eur. J. Neurosci., 29, 1842_1852, 2009に記載の方法に準じて行った。
Example 2 Pharmacological Test Next, the method and results of a pharmacological test regarding the L-PGDS production promoting effect using the present extract obtained in Example 1 above as a test substance will be shown. In addition, in the following pharmacological test, regarding the introduction of cerebral infarction into CB-17 mice and the isolation and culture of iSC from the cerebral infarct focus, please refer to Nakagomi, T. et al. Eur. J. Neurosci., 29, 1842_1852, 2009.
試験例１：被験物質処理iSCにおける網羅的遺伝子発現解析
C.B-17マウス（3匹）に中大脳動脈閉塞による虚血負荷を施し、3日目に梗塞巣より3株のiSCを単離した。それぞれの培養iSC（2% ウシ胎児血清（FBS）、20ng/mL 線維芽細胞増殖因子（FBS）、20ng/mL 上皮成長因子（EGF）及び1％ N2サプリメントを添加したダルベッコ改変イーグル培地F12（2% FBS DMEM/F12 F/E/N）、5×104 cell/3cmφdish）に対して被験物質（50、1000 mNU/mL）又は生理食塩液（コントロール）を添加し、培養4日目にRNeasy〔登録商標、以下同様〕 Mini Kit（QIAGEN社）により全RNAを回収した（全9培養）。網羅的遺伝子発現解析には、マウス用の遺伝子チップSurePrint G3 Mouse GE マイクロアレイ8×60K（24,321 種類のRNA及び4,576種類のノンコーディングRNA、GE社）を用い、被験物質の添加によって3株ともに両濃度で同方向に1/2以下の低下ないし2倍以上の発現変化を示す遺伝子を選別した。結果は、コントロールに対する選別した各遺伝子の発現量比として表した（3株の平均値±標準誤差）。結果の一例を表１に示す。
Test Example 1: Comprehensive gene expression analysis in test substance-treated iSCs
CB-17 mice (3 mice) were subjected to ischemic stress by occlusion of the middle cerebral artery, and on the 3rd day, 3 strains of iSC were isolated from the infarcted lesion. Cultured iSCs (2% Fetal Bovine Serum (FBS), 20ng/mL Fibroblast Growth Factor (FBS), 20ng/mL Epidermal Growth Factor (EGF) and 1% N2 supplemented in Dulbecco's Modified Eagle Medium F12 (2 % FBS DMEM/F12 F/E/N), 5×10 4 cells/3cmφdish), add the test substance (50, 1000 mNU/mL) or physiological saline (control), and on the 4th day of culture, add RNeasy. [Registered trademark, same hereinafter] Total RNA was collected using Mini Kit (QIAGEN) (9 cultures in total). For comprehensive gene expression analysis, a mouse gene chip SurePrint G3
上記解析の結果、表１に示す通り、発現減少を示したCOCH遺伝子並びに発現上昇を示したGBP6遺伝子及びPTGDS遺伝子の計3遺伝子が選別された。これらのうち、L-PGDSをコードするPTGDS遺伝子の発現量は被験物質に対する強い用量依存性を示した。 As a result of the above analysis, as shown in Table 1, a total of three genes were selected: the COCH gene, which showed decreased expression, and the GBP6 gene and PTGDS gene, which showed increased expression. Among these, the expression level of the PTGDS gene encoding L-PGDS showed a strong dose dependence on the test substance.
試験例２－１：L-PGDS遺伝子発現に対する評価（リアルタイムRT-PCR法）
試験例１と同様に、培養iSC（FBS不含 DMEM/F12 F/-/-、5×104 cell/3cmφdish）に被験物質（50、500 mNU/mL）又は生理食塩液（コントロール）を添加し（n = 1）、 7日間培養した後，Isogen II〔登録商標〕により製造元（ニッポンジーン社）のマニュアルに従ってRNAを回収した。260 nm及び280 nmの吸光度を指標にRNA純度を検定した後、ランダムプライマー存在下でSuperScript〔登録商標、以下同様〕IV RTase（Invitrogen社）による逆転写反応を行って、全RNAに対する一本鎖cDNAを得た。得られたcDNAについて、PTGDS特異的プライマーを用いた定量的PCR法（Prism〔登録商標〕7900HT、Applied Biosystems社）により、ハウスキーピング遺伝子β-Actinを標準として転写産物の定量を行った（閾値リサイクル比較法）。なお、使用したPTGDS及びβ-Actin用プライマーの配列は、以下の通りである〔PTGDS：5'-gactctgaaggacgagctgaag-3'（配列番号１）、5'-tcttgaatgcacttatccggttgg-3'（配列番号２）、・-Actin：5'-tacagcttcaccaccacagc-3'（配列番号３）、5'-aaggaaggctggaaaagagc-3'（配列番号４）〕。結果は、コントロールを1とした時の・-Actinに対するPTGDSの発現量比で示した。結果の一例を表２及び図１に示す。
Test Example 2-1: Evaluation of L-PGDS gene expression (real-time RT-PCR method)
As in Test Example 1, add the test substance (50, 500 mNU/mL) or physiological saline (control) to cultured iSCs (DMEM/F12 F/-/- without FBS, 5 × 10 4 cells/3 cmφ dish). After culturing for 7 days (n = 1), RNA was collected using Isogen II (registered trademark) according to the manufacturer's manual (Nippon Gene). After assaying RNA purity using absorbance at 260 nm and 280 nm as indicators, reverse transcription reaction using SuperScript (registered trademark, hereinafter the same) IV RTase (Invitrogen) was performed in the presence of random primers to obtain single-stranded RNA from total RNA. cDNA was obtained. The transcripts of the obtained cDNA were quantified by quantitative PCR using PTGDS-specific primers (Prism (registered trademark) 7900HT, Applied Biosystems) using the housekeeping gene β-Actin as a standard (threshold recycling). comparative law). The sequences of the PTGDS and β-Actin primers used are as follows [PTGDS: 5'-gactctgaaggacgagctgaag-3' (SEQ ID NO: 1), 5'-tcttgaatgcacttatccggttgg-3' (SEQ ID NO: 2), -Actin: 5'-tacagcttcaccaccacagc-3' (SEQ ID NO: 3), 5'-aaggaaggctggaaaagagc-3' (SEQ ID NO: 4)]. The results were expressed as the expression level ratio of PTGDS to ・-Actin when the control was set to 1. An example of the results is shown in Table 2 and FIG. 1.
表２及び図１から明らかなように、試験例１で示された結果と同様に、被験物質はiSCにおいてL-PGDS遺伝子（PTGDS）の発現を用量依存的に促進することが確認された。 As is clear from Table 2 and FIG. 1, similar to the results shown in Test Example 1, it was confirmed that the test substance promoted the expression of the L-PGDS gene (PTGDS) in iSCs in a dose-dependent manner.
試験例２－２：L-PGDS遺伝子発現に対する評価（古典的RT-PCR法）
0（コントロール；生理食塩液）、1、5、50、100 mNU/mLの各用量の被験物質存在下、FGFを含有する培地（DMEM/F12 F/-/-）又はFGFを含有しない培地（DMEM/F12 -/-/-）で、iSCを4日間培養した。RNeasy Mini Kit（QIAGEN社）により回収された全RNAに対して、SuperScript III One-Step RT-PCR System with Platinum（Invitrogen社）を用いた古典的RT-PCR法によってL-PGDS遺伝子（PTGDS）の発現量を半定量した（35サイクル）。PCR産物を2%アガロースゲル電気泳動により分離し、PTGDS及びGAPDHのバンドを臭化エチジウムで染色して可視的に検出した。なお、使用したPTGDS及びGAPDH用プライマーの配列は、以下の通りである〔PTGDS：5'-cctccaactcaagctggttc-3'（配列番号５）、5'-atagttggcctccaccactg-3'（配列番号６）、GAPDH：5'-atcactgccacccagaagac-3'（配列番号７）、5'-cacattgggggtaggaacac-3'（配列番号８）〕。結果の一例を図２に示す。
Test Example 2-2: Evaluation of L-PGDS gene expression (classical RT-PCR method)
In the presence of the test substance at doses of 0 (control; physiological saline), 1, 5, 50, and 100 mNU/mL, a medium containing FGF (DMEM/F12 F/-/-) or a medium without FGF ( iSCs were cultured in DMEM/F12 -/-/- for 4 days. L-PGDS gene (PTGDS) was isolated by classical RT-PCR using SuperScript III One-Step RT-PCR System with Platinum (Invitrogen) on total RNA recovered with RNeasy Mini Kit (QIAGEN). Expression levels were semi-quantified (35 cycles). PCR products were separated by 2% agarose gel electrophoresis, and PTGDS and GAPDH bands were visually detected by staining with ethidium bromide. The sequences of the PTGDS and GAPDH primers used are as follows [PTGDS: 5'-cctccaactcaagctggttc-3' (SEQ ID NO: 5), 5'-atagttggcctccaccactg-3' (SEQ ID NO: 6), GAPDH: 5 '-atcactgccacccagaagac-3' (SEQ ID NO: 7), 5'-cacattgggggtaggaacac-3' (SEQ ID NO: 8)]. An example of the results is shown in FIG. 2.
iSCにおけるL-PGDS遺伝子（PTGDS）発現促進作用について、より細かい被験物質の用量で調べたところ、図２に示される通り、被験物質は、培地のFGF含有の有無に関わらず、L-PGDS遺伝子の発現量を用量依存的に増加させた。 The effect of promoting L-PGDS gene (PTGDS) expression in iSCs was investigated using more detailed doses of the test substance. As shown in Figure 2, the test substance was effective at promoting L-PGDS gene expression in iSCs, regardless of whether the medium contained FGF or not. The expression level of was increased in a dose-dependent manner.
試験例３－１：L-PGDSタンパクの発現に対する評価（ウエスタンブロッティング法）
iSCを血清非存在下で被験物質（0、1、5、50、100 mNU/mL）を添加して4日間培養（DMEM/F12 F/-/-、5×104 cell/3cmφdish）した後回収し、リン酸緩衝液で洗浄後、RIPA緩衝液（4℃、50 mM トリス塩酸緩衝液（pH 7.6)、150 mM 塩化ナトリウム、1% ノニデット〔登録商標〕P-40（NP-40）、0.5% デオキシコール酸ナトリウム及び0.1% ドデシル硫酸ナトリウム）により溶解し、ホモジネートとした。総タンパク量が均一となるように調整後、ホモジネートをSDS-PAGE（BIO-RAD Any kD（商標））で分離し、ポリフッ化ビニリデン（PVDF）膜（Immun-Blot PVDF メンブレン、BIO-RAD社）に転写して、Bloking One（nacalai tesque社）でブロッキング後、特異的な抗体を用いたウエスタンブロッティング法によりL-PGDS（anti-Prostaglandin D Synthase（Lipocalin）antibody [EP12357]、Abcam社、1:2000）及びβ-Actin（Monoclonal anti-β-Actin [A1978]、Sigma社、1:100000）を検出した。検出には高感度化学発光法（Chemi-Lumi One L, nacalai tesque 社）を使用した。結果の一例を図３に示す。
Test Example 3-1: Evaluation of L-PGDS protein expression (Western blotting method)
After iSCs were cultured for 4 days (DMEM/F12 F/-/-, 5×10 4 cells/3cmφdish) with the addition of the test substance (0, 1, 5, 50, 100 mNU/mL) in the absence of serum. After collecting and washing with phosphate buffer, RIPA buffer (4℃, 50 mM Tris-HCl buffer (pH 7.6), 150 mM sodium chloride, 1% Nonidet [registered trademark] P-40 (NP-40), 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) to make a homogenate. After adjusting the total protein content to be uniform, the homogenate was separated by SDS-PAGE (BIO-RAD Any kD (trademark)) and separated using a polyvinylidene fluoride (PVDF) membrane (Immun-Blot PVDF membrane, BIO-RAD). After blocking with Blocking One (Nacalai Tesque), L-PGDS (anti-Prostaglandin D Synthase (Lipocalin) antibody [EP12357], Abcam, 1:2000) was transferred using Western blotting using a specific antibody. ) and β-Actin (Monoclonal anti-β-Actin [A1978], Sigma, 1:100000) were detected. A highly sensitive chemiluminescence method (Chemi-Lumi One L, Nacalai Tesque) was used for detection. An example of the results is shown in FIG.
図３から明らかなように、被験物質はiSC細胞内のL-PGDSタンパクの発現を促進させることが確認された。 As is clear from FIG. 3, it was confirmed that the test substance promoted the expression of L-PGDS protein in iSC cells.
試験例３－２：L-PGDSタンパクの発現に対する評価（ドットブロッティング法）
被験物質（0、1、10、50、100 mNU/mL）で4日間培養（DMEM/F12 F/-/-、5×104 cell/3cmφdish）したiSCの培養上清500μLをPVDF膜（Immun-Blot PVDF メンブレン、BIO-RAD社）にスポットし、Bloking One （nacalai tesque社）でブロッキング後、抗L-PGDS抗体（anti-Prostaglandin D Synthase（Lipocalin）antibody [EP12357]、Abcam社、1：2000）を用いたドットブロッティング法により、L-PGDSを検出した。結果の一例を図４に示す。
Test Example 3-2: Evaluation of L-PGDS protein expression (dot blotting method)
500 μL of the culture supernatant of iSCs cultured for 4 days (DMEM/F12 F/-/-, 5×10 4 cells/3 cmφ dish) with the test substance (0, 1, 10, 50, 100 mNU/mL) was transferred to a PVDF membrane (Immun -Blot PVDF membrane, BIO-RAD) and after blocking with Blocking One (Nacalai Tesque), anti-L-PGDS antibody (anti-Prostaglandin D Synthase (Lipocalin) antibody [EP12357], Abcam, 1:2000 ) was used to detect L-PGDS. An example of the results is shown in FIG.
L-PGDSはそのN末端に典型的な分泌シグナル配列及びシグナルペプチダーゼ認識配列を有することから、細胞外へ分泌されると考えられる。そこで、被験物質を添加したiSCの培養上清中のL-PGDSタンパクを測定したところ、図４から明らかなように、培養上清中（細胞外）のL-PGDSタンパク量も被験物質の添加用量に依存して増加することが確認された。 Since L-PGDS has a typical secretion signal sequence and signal peptidase recognition sequence at its N-terminus, it is thought to be secreted outside the cell. Therefore, when we measured L-PGDS protein in the culture supernatant of iSCs to which the test substance had been added, as is clear from Figure 4, the amount of L-PGDS protein in the culture supernatant (extracellularly) also increased after the addition of the test substance. It was confirmed that the increase was dose-dependent.
試験例３－３：L-PGDSタンパク質の発現に対する評価（ELISA法）
被験物質（0〔コントロール〕、1、10、50、100 、1000mNU/mL）で4日間処理したiSCの培養（DMEM/F12 F/-/-、5×104 cell/3cmφdish）の上清を回収した。遠心分離（1500rpm、10分、4℃）後、培養上清中のL-PGDS量を特異的ELISA法（ヒトL-PGDS用 ELISA キット（Prostaglandin D Synthase 21kDa (Brain)、型番：SEA724Hu、Cloud-Clone Corp.）を用い、製造者のマニュアルに従って測定した。結果の一例を表３及び図５に示す。
Test Example 3-3: Evaluation of L-PGDS protein expression (ELISA method)
The supernatant of iSC culture (DMEM/F12 F/-/-, 5×10 4 cells/3cmφdish) treated with the test substance (0 [control], 1, 10, 50, 100, 1000 mNU/mL) for 4 days was Recovered. After centrifugation (1500 rpm, 10 minutes, 4℃), the amount of L-PGDS in the culture supernatant was measured using a specific ELISA method (ELISA kit for human L-PGDS (Prostaglandin D Synthase 21kDa (Brain), model number: SEA724Hu, Cloud- Clone Corp.) according to the manufacturer's manual. An example of the results is shown in Table 3 and FIG.
表３及び図５から明らかなように、試験例３－２と同様に、被験物質で処理したiSCの培養上清（細胞外）において、L-PGDSタンパク量が増加することが確認された。試験例３の結果から、被験物質は、iSCにおいて、遺伝子レベルのみならず、タンパクレベルにおいてもL-PGDSの産生を促進し、さらに、産生されたL-PGDSを分泌させることが認められた。 As is clear from Table 3 and FIG. 5, similarly to Test Example 3-2, it was confirmed that the amount of L-PGDS protein increased in the culture supernatant (extracellular) of iSCs treated with the test substance. From the results of Test Example 3, it was observed that the test substance promoted the production of L-PGDS in iSCs not only at the gene level but also at the protein level, and further caused the produced L-PGDS to be secreted.
試験例４：L-PGDSの酵素活性に対する評価（液体クロマトグラフィー（HPLC）質量分析法）
L-PGDSは、PGH2を基質としてPGD2を生合成する酵素（EC 5.3.99.2）である。iSCにおいて、被験物質によって産生促進されたL-PGDSが、その酵素活性によりさらにPGD2を産生することを確認する目的で、iSCのA：細胞内及びB：細胞外において、PGD2及びその代謝産物、並びにPGD2と同じくPGH2を基質として産生されるPGE2等のプロスタグランジン類を網羅的に分析した。
Test Example 4: Evaluation of enzyme activity of L-PGDS (liquid chromatography (HPLC) mass spectrometry)
L-PGDS is an enzyme (EC 5.3.99.2) that biosynthesizes PGD2 using PGH2 as a substrate. In order to confirm that L-PGDS whose production was promoted by the test substance in iSCs further produces PGD2 through its enzymatic activity, PGD2 and its metabolites, In addition, prostaglandins such as PGE2, which are produced using PGH2 as a substrate like PGD2, were comprehensively analyzed.
A：iSC細胞内酵素活性の評価
被験物質（0、10、50 mNU/mL）を添加して3日間培養（FBS不含 DMEM/F12 F/-/-、5×104 cell/3cmφ dish）したiSCから、RIPA緩衝液処理（4℃、20 min）により細胞抽出液を調製した。HPLC質量分析法により、細胞内抽出液中のプロスタグランジン類（PGD2、PGJ2、15-デオキシ-Δ12,14-PGJ2、13,14-ジヒドロ-15-ケト-PGD2及びPGE2）の濃度を各々測定した。HPLC分離には、AQUITY UPLC HSS T3カラム（Waters社）を用い、検出器及び質量分析機器として各々API4000 LC/MS/MSシステム及びTriple Quadrupole（いずれもAB Sciex社）を使用した。結果の一例を表４及び図６に示す。
A: Evaluation of iSC intracellular enzyme activity Test substance (0, 10, 50 mNU/mL) was added and cultured for 3 days (DMEM/F12 F/-/- without FBS, 5×10 4 cells/3cmφ dish) A cell extract was prepared from the iSCs by treatment with RIPA buffer (4°C, 20 min). Measure the concentration of prostaglandins (PGD2, PGJ2, 15-deoxy-Δ12,14-PGJ2, 13,14-dihydro-15-keto-PGD2 and PGE2) in intracellular extracts by HPLC mass spectrometry. did. For HPLC separation, an AQUITY UPLC HSS T3 column (Waters) was used, and an API4000 LC/MS/MS system and Triple Quadrupole (both AB Sciex) were used as a detector and a mass spectrometer, respectively. An example of the results is shown in Table 4 and FIG. 6.
表４及び図６に示される通り、PGD2及びその非酵素的代謝産物であるPGJ2の細胞内レベルは被験物質添加により用量依存的に増加した。さらに、50 mNU/mLでの被験物質添加においては、PGJ2の非酵素的代謝産物である15-デオキシ-Δ12,14-PGJ2の産生も確認された。なお、15-デオキシ-Δ12,14-PGJ2の非酵素的代謝産物である13,14-ジヒドロ-15-ケト-PGD2は検出限界以下であった。一方で、PGD2と共通の基質PGH2から産生されるPGE2については、被験物質添加の用量に依存した産生応答を観察できなかった。 As shown in Table 4 and FIG. 6, the intracellular levels of PGD2 and its non-enzymatic metabolite PGJ2 increased in a dose-dependent manner upon addition of the test substance. Furthermore, when the test substance was added at 50 mNU/mL, production of 15-deoxy-Δ12,14-PGJ2, a non-enzymatic metabolite of PGJ2, was also confirmed. Note that 13,14-dihydro-15-keto-PGD2, a non-enzymatic metabolite of 15-deoxy-Δ12,14-PGJ2, was below the detection limit. On the other hand, for PGE2 produced from the common substrate PGH2 with PGD2, no dose-dependent production response was observed when the test substance was added.
B：iSC細胞外酵素活性の評価
iSCを被験物質（0、50、100、1000 mNU/mL）を添加して3日間培養（FBS不含 DMEM/F12 F/-/-、5×104 cell/3cmφ dish）した培養上清に、L-PGDSの基質であるPGH2及びグルタチオン（GSH）を作用させた（反応条件：100 mM Tris-HCl (pH8.0)、1 mM GSH、10 mM PGH2、37℃、5 min）。上記A．と同様に、HPLC質量分析法により、反応液中のプロスタグランジン類（PGD2、PGJ2、15-デオキシ-Δ12,14-PGJ2、13,14-ジヒドロ-15-ケト-PGD2及びPGE2）の濃度を各々測定した。結果の一例を表５及び図７に示す。
B: Evaluation of iSC extracellular enzyme activity
iSCs were added to the test substance (0, 50, 100, 1000 mNU/mL) and cultured for 3 days (FBS-free DMEM/F12 F/-/-, 5×10 4 cells/3cmφ dish) in the culture supernatant. , PGH2, which is a substrate of L-PGDS, and glutathione (GSH) were reacted (reaction conditions: 100 mM Tris-HCl (pH 8.0), 1 mM GSH, 10 mM PGH2, 37°C, 5 min). Above A. Similarly, the concentration of prostaglandins (PGD2, PGJ2, 15-deoxy-Δ12,14-PGJ2, 13,14-dihydro-15-keto-PGD2 and PGE2) in the reaction solution was determined by HPLC mass spectrometry. Each was measured. An example of the results is shown in Table 5 and FIG. 7.
表５及び図７に示される通り、被験物質は100 mNU/mL以上の濃度で反応液中のPGD2及びその非酵素的代謝産物PGJ2、15-デオキシ-Δ12,14-PGJ2の濃度を上昇させたことから、iSC培養上清中にL-PGDS活性が存在することが確認された。また、15-デオキシ-Δ12,14-PGJ2の非酵素的代謝産物である13,14-ジヒドロ-15-ケト-PGD2については、100 mNU/mLまでの濃度範囲の被験物質添加により用量に依存した産生を観察したが、1000 mNU/mLにおいては検出限界を下回った。 As shown in Table 5 and Figure 7, the test substance increased the concentration of PGD2 and its non-enzymatic metabolites PGJ2 and 15-deoxy-Δ12,14-PGJ2 in the reaction solution at concentrations of 100 mNU/mL or higher. This confirmed the presence of L-PGDS activity in the iSC culture supernatant. In addition, for 13,14-dihydro-15-keto-PGD2, a non-enzymatic metabolite of 15-deoxy-Δ12,14-PGJ2, addition of the test substance at concentrations up to 100 mNU/mL caused a dose-dependent effect. Although production was observed, it was below the detection limit at 1000 mNU/mL.
L-PGDSは分泌型酵素であることが知られているが、上記A．及びB．の結果から、iSCは酵素活性を有するL-PGDSを細胞外へ放出しており、被験物質添加によりその産生と放出が促進されることが確認された。 L-PGDS is known to be a secreted enzyme, but the above A. and B. From the results, it was confirmed that iSCs release L-PGDS with enzymatic activity to the outside of the cells, and that addition of the test substance promoted its production and release.
試験例５－１：マウス脳内におけるL-PGDS産生細胞の検討（免疫組織化学染色法）
中大脳動脈閉塞による虚血負荷を施したC.B-17マウス（虚血3日後）の脳切片を作製し、L-PGDS（パネルB、緑；anti-prostaglandin D synthase (lipocalin) antibody [EP12357]、Abcam社、1:1000）、ペリサイトマーカーα-SMA（パネルC、赤；anti-actin、smooth muscle、clone ASM-1、Millipore社、1：1000）及び血管内皮細胞マーカーCD31（パネルD、赤；anti-mouse CD31 (PECAM-1) monoclonal antibody、550274、BD Pharmingen社、1:1000）に対する特異的抗体を用いて、共焦点レーザー顕微鏡による免疫組織化学的検討を行った。多重染色法により、L-PGDS（パネルB）とペリサイトマーカー（パネルC）又はL-PGDS（パネルB）と血管内皮細胞マーカー（パネルD）を各々異なる蛍光色素で標識された特異的抗体で検出し、画像を重ね合わせて、L-PGDSとペリサイトマーカー又はL-PGDSと血管内皮細胞マーカーの分布について比較した（パネルA1、パネルA2）。結果の一例を図８に示す。
Test Example 5-1: Examination of L-PGDS producing cells in mouse brain (immunohistochemical staining method)
Brain sections of CB-17 mice (3 days after ischemia) subjected to ischemic stress due to middle cerebral artery occlusion were prepared and treated with L-PGDS (panel B, green; anti-prostaglandin D synthase (lipocalin) antibody [EP12357]), Abcam, 1:1000), pericyte marker α-SMA (panel C, red; anti-actin, smooth muscle, clone ASM-1, Millipore, 1:1000) and vascular endothelial cell marker CD31 (panel D, red). ; anti-mouse CD31 (PECAM-1) monoclonal antibody, 550274, BD Pharmingen, 1:1000), immunohistochemical examination was performed using a confocal laser microscope. Using multiple staining, L-PGDS (Panel B) and pericyte markers (Panel C) or L-PGDS (Panel B) and vascular endothelial cell markers (Panel D) were each labeled with specific antibodies labeled with different fluorescent dyes. were detected, the images were superimposed, and the distributions of L-PGDS and pericyte markers or L-PGDS and vascular endothelial cell markers were compared (panel A1, panel A2). An example of the results is shown in FIG.
図８上段において、パネルBの緑色蛍光はL-PGDSの分布を示し、パネルCの赤色蛍光はペリサイトマーカーの分布を示すが、両者を重ね合わせたパネルＡ1において、L-PGDS（緑色蛍光）はペリサイトマーカー（赤色蛍光）の分布と部分的に一致した（200倍）。また、図８下段において、パネルBの緑色蛍光はL-PGDSの分布を示し、パネルDの赤色蛍光は血管内皮細胞マーカーの分布を示すが、両者を重ね合わせたパネルＡ2において、L-PGDS（緑色蛍光）は血管内皮細胞マーカー（赤色蛍光）の近傍に分布するものの、共分布を示す観察は得られなかった（200倍）。 In the upper part of Figure 8, green fluorescence in panel B indicates the distribution of L-PGDS, and red fluorescence in panel C indicates the distribution of pericyte markers. partially matched the distribution of pericyte markers (red fluorescence) (200x magnification). In addition, in the lower part of Figure 8, green fluorescence in panel B indicates the distribution of L-PGDS, and red fluorescence in panel D indicates the distribution of vascular endothelial cell markers. Although the vascular endothelial cell marker (green fluorescence) was distributed near the vascular endothelial cell marker (red fluorescence), no observation indicating co-distribution was obtained (200x magnification).
L-PGDSは主に中枢神経系に分布するタンパクであり、クモ膜や軟膜及び側脳室の脈絡叢などに存在し、脳脊髄液中に分泌されると考えられている（Urade Y. et al., J Lipid Mediat. Cell Signal. 14, 71-82, 1996）が、脳内産生細胞については十分に解明されていない。本発明者らは、免疫組織化学的手法によって、L-PGDSの脳内分布に関する検討を行ったところ、C.B-17マウスの正常脳における発現は微弱であったが、上記試験例５－１において、本マウスに中大脳動脈の永久結紮を導入すると、脳梗塞巣に顕著なL-PGDS発現が認められた（図８のパネルB）。また、L-PGDSの分布は血管内皮細胞のマーカーであるCD31の周囲に認められたが、両者の分布が重なることはなかった（図８のパネルA2）。一方、L-PGDSはペリサイトのマーカーであるα-SMAと一部共分布を示すことが分かった（図８のパネルA1）。以上の結果より、脳梗塞巣において発現誘導されるL-PGDSは、ペリサイト（あるいはiSC）に由来するものであると考えられた。このことは、後述する試験例５－２の免疫電子顕微鏡法によっても、L-PGDS陽性産物が血管内皮細胞と基底膜に接して存在するペリサイトの細胞質内に電子密度の高い構造物として確認されたことにより検証された（図９のパネルA及びB）。これらの結果より、虚血応答におけるペリサイト又はiSCの生理学的役割として、L-PGDSの発現誘導が含まれると想像された。 L-PGDS is a protein mainly distributed in the central nervous system, present in the arachnoid mater, pia mater, and choroid plexus of the lateral ventricle, and is thought to be secreted into the cerebrospinal fluid (Urade Y. et al. al., J Lipid Mediat. Cell Signal. 14, 71-82, 1996), but the producing cells in the brain are not fully understood. The present inventors investigated the distribution of L-PGDS in the brain using immunohistochemical techniques, and found that the expression in the normal brain of C.B-17 mice was weak; When permanent ligation of the middle cerebral artery was introduced into these mice, significant L-PGDS expression was observed in the cerebral infarction focus (panel B of Figure 8). Furthermore, although the distribution of L-PGDS was observed around CD31, which is a marker for vascular endothelial cells, the two distributions did not overlap (panel A2 of FIG. 8). On the other hand, L-PGDS was found to partially co-distribute with α-SMA, a pericyte marker (panel A1 in Figure 8). From the above results, it was considered that L-PGDS, whose expression is induced in cerebral infarction foci, is derived from pericytes (or iSCs). This was also confirmed by immunoelectron microscopy in Test Example 5-2, which will be described later, as L-PGDS-positive products were confirmed as electron-dense structures within the cytoplasm of pericytes that are present in contact with vascular endothelial cells and the basement membrane. (Panels A and B of Figure 9). From these results, it was assumed that the physiological role of pericytes or iSCs in the ischemic response includes induction of L-PGDS expression.
試験例５－２：マウス脳内におけるL-PGDS産生細胞の検討（免疫電子顕微鏡法）
中大脳動脈閉塞による虚血負荷を施したC.B-17マウス（虚血3日後）の脳切片を作製し、L-PGDS（anti-prostaglandin D synthase (lipocalin) antibody [EP12357]、Abcam社、1：1000）に対する特異的抗体を用いた免疫電子顕微鏡法により、L-PGDSの発現部位を観察した。具体的には、ビブラトームにて2μm厚の脳切片を作製し、avidin-biotin horseradish peroxidase (HRP) complex キット（Vector Laboratories）と 3,3'-diaminobenzidinetetrahydrochloride （DAB）を用いた反応後、オスミウム処理を行い、エポン包埋後、超薄切片を作製して電顕観察を行った。結果の一例を図９に示す。
Test Example 5-2: Examination of L-PGDS producing cells in mouse brain (immunoelectron microscopy)
Brain sections of CB-17 mice (3 days after ischemia) subjected to ischemic stress due to middle cerebral artery occlusion were prepared and treated with L-PGDS (anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1: The expression site of L-PGDS was observed by immunoelectron microscopy using a specific antibody against 1000). Specifically, 2 μm thick brain sections were prepared using a vibratome, reacted with the avidin-biotin horseradish peroxidase (HRP) complex kit (Vector Laboratories) and 3,3'-diaminobenzidinetetrahydrochloride (DAB), and then treated with osmium. After embedding in Epon, ultrathin sections were prepared and observed with an electron microscope. An example of the results is shown in FIG.
図９のパネルA及びBのいずれにおいても、L-PGDSに対する免疫電子顕微鏡法では、血管内皮細胞と基底膜に接して存在する血管周皮細胞（ペリサイト）の細胞質内に電子密度の高い領域が確認された。 In both panels A and B of Figure 9, immunoelectron microscopy for L-PGDS reveals regions with high electron density within the cytoplasm of vascular endothelial cells and vascular pericytes (pericytes) that exist in contact with the basement membrane. was confirmed.
試験例６：ヒト脳内におけるL-PGDS産生細胞の検討（免疫組織化学染色法）
Tatebayashi K. et al., Stem Cells and Develop. 26, 787-797, 2017に記載の方法に準じて、ヒト脳梗塞巣（壊死組織）から抽出した培養 iSC において、L-PGDS（パネルB、緑；anti-prostaglandin D synthase (lipocalin) antibody [EP12357]、Abcam社、1:1000）と神経幹細胞マーカーnestin（パネルC、赤；anti-nestin、clone 10C2、Millipore社、1:1000）に対する特異的抗体を用いた免疫組織化学的検討を行った。細胞は、Alexa Fluor〔登録商標、以下同様〕488-conjugated抗体又はAlexa Fluor 555-conjugated抗体（1:500；Molecular Probes、Eugene）と反応後に4',6-diamidino-2-phenylindole （DAPI；1:1000；Kirkegaard & Perry Laboratories社）による核染色をおこなった。Fluorescent microscope （BX60；Olympus，Japan）で蛍光観察し、L-PGDS（パネルB）とnestin（パネルC）の画像を重ね合わせて、L-PGDSとnestinの分布について比較した（パネルA）。結果の一例を図１０に示す。
Test Example 6: Examination of L-PGDS producing cells in human brain (immunohistochemical staining method)
According to the method described in Tatebayashi K. et al., Stem Cells and Develop. 26, 787-797, 2017, L-PGDS (panel B, green ; anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1:1000) and neural stem cell marker nestin (Panel C, red; anti-nestin, clone 10C2, Millipore, 1:1000) Immunohistochemical studies were performed using After reacting with Alexa Fluor (registered trademark) 488-conjugated antibody or Alexa Fluor 555-conjugated antibody (1:500; Molecular Probes, Eugene), cells were treated with 4',6-diamidino-2-phenylindole (DAPI; :1000; Nuclear staining was performed using Kirkegaard & Perry Laboratories). Fluorescence was observed using a fluorescent microscope (BX60; Olympus, Japan), and the images of L-PGDS (panel B) and nestin (panel C) were superimposed to compare the distributions of L-PGDS and nestin (panel A). An example of the results is shown in FIG.
図１０において、パネルBの緑色蛍光はL-PGDSの分布を示し、パネルCの赤色蛍光はnesitinの分布を示すが、両者を重ね合わせたパネルAでは、L-PGDS（緑色蛍光）はnestin（赤色蛍光）の分布と一致した（200倍）。すなわち、L-PGDSはnestinを発現しているほぼすべてのiSCで発現することが確認された。以上より、マウス脳のみならず、ヒト脳においてもL-PGDSはiSCあるいはペリサイトで産生されると考えられた。 In Figure 10, the green fluorescence in panel B shows the distribution of L-PGDS, and the red fluorescence in panel C shows the distribution of nestin, but in panel A, in which the two are superimposed, L-PGDS (green fluorescence) shows the distribution of nestin ( (red fluorescence) distribution (200x magnification). In other words, it was confirmed that L-PGDS is expressed in almost all iSCs expressing nestin. From the above, it was considered that L-PGDS is produced in iSCs or pericytes not only in the mouse brain but also in the human brain.
試験例７：アルツハイマー型認知症モデル動物の脳内アミロイドβ沈着とL-PGDS発現に対する評価
（１）認知能に関する行動学的解析及びサンプル調製
APPswe/PS1dE9（APP/PS1）マウス（雌性3ヶ月齢）を各群体重が均等になるように被験物質投与群とコントロール群に分け（各群10～11匹）、約3ヶ月間にわたり各々被験物質（100 NU/kg体重）又は生理食塩液を週2回、尾静注した。最終投与後、認知能に関する行動学的解析（Y字迷路試験、新奇物体認識試験及びモリス水迷路試験）を行った。各試験において、A：Y字迷路試験では交替行動率（％）、B：新奇物体認識試験では新奇物体に対するアクセス頻度（％）、及びC：モリス水迷路試験では学習期間の終了時までの4日間（6～9日目）のプラットホーム発見に要する平均時間（秒）を各々測定し、個体ごとに統合スコア（A×B÷C）を算出した。各群2個体ずつの測定結果を表６に示す。
Test Example 7: Evaluation of intracerebral amyloid β deposition and L-PGDS expression in Alzheimer's disease model animals (1) Behavioral analysis of cognitive ability and sample preparation
APPswe/PS1dE9 (APP/PS1) mice (female, 3 months old) were divided into a test substance administration group and a control group (10 to 11 mice in each group) so that each group's body weight was equal, and each group was subjected to the test substance for approximately 3 months. Substances (100 NU/kg body weight) or saline were injected into the tail vein twice a week. After the final administration, behavioral analyzes regarding cognitive ability (Y-maze test, novel object recognition test, and Morris water maze test) were performed. In each test, A: rate of alternation behavior (%) in the Y-maze test, B: frequency of access to the novel object (%) in the novel object recognition test, and C: 4 by the end of the learning period in the Morris water maze test. The average time (seconds) required to discover the platform for each day (
表６から明らかなように、被験物質投与群による認知能の改善作用が観察され、この作用は群間では統計学的に有意であった。
認知能に関する行動学的解析後（最終投与24時間後）、被験物質投与群及びコントロール群において、麻酔下断頭により脳を採取し、左半球を液体窒素バスにて急速凍結した。また、残りの右半球は、（A）グルタールアルデヒドを含む固定液（0.05％グルタールアルデヒド、4％パラフォルムアルデヒド、0.1Mリン酸バッファー）又は（B）PLP固定液（0.01Mメタ過ヨウ素酸ナトリウム、0.075Mリジン、2％パラフォルムアルデヒド）により固定した（4℃）。固定組織標本からクライオスタット（Leica CM1850）により大脳皮質を含む冠状面切片（厚さ20μm）を作製した。
As is clear from Table 6, an effect of improving cognitive ability was observed depending on the test substance administration group, and this effect was statistically significant between the groups.
After behavioral analysis of cognitive ability (24 hours after the final administration), the brains of the test substance administered group and the control group were collected by decapitation under anesthesia, and the left hemisphere was rapidly frozen in a liquid nitrogen bath. In addition, the remaining right hemisphere was treated with (A) fixative containing glutaraldehyde (0.05% glutaraldehyde, 4% paraformaldehyde, 0.1M phosphate buffer) or (B) PLP fixative (0.01M metaperiodide). (sodium chloride, 0.075 M lysine, 2% paraformaldehyde) (4°C). A coronal section (20 μm thick) containing the cerebral cortex was prepared from the fixed tissue specimen using a cryostat (Leica CM1850).
（２）大脳皮質におけるアミロイドβ沈着に対する評価（免疫組織化学染色法）
上記（１）で作製した被験物質投与群及びコントロール群の冠状面切片について、抗アミロイドβ抗体（anti-β-amyloid、1-16、[SIG-39300]、BioLegend社、1:1000）による免疫組織化学的染色を行った。アミロイドβは、Alexa Fluor 488-conjugated抗体（1:500；Molecular Probes、Eugene）と反応後に4',6-diamidino-2-phenylindole（DAPI；1:1000；Kirkegaard & Perry Laboratories）による核染色を行い、Fluorescent microscope（BX60；Olympus, Japan）で蛍光観察した。結果の一例を図１１に示す。
(2) Evaluation of amyloid β deposition in the cerebral cortex (immunohistochemical staining method)
Immunization with anti-amyloid-β antibody (anti-β-amyloid, 1-16, [SIG-39300], BioLegend, 1:1000) for the coronal sections of the test substance administration group and control group prepared in (1) above. Histochemical staining was performed. Amyloid β was detected by nuclear staining with 4',6-diamidino-2-phenylindole (DAPI; 1:1000; Kirkegaard & Perry Laboratories) after reacting with Alexa Fluor 488-conjugated antibody (1:500; Molecular Probes, Eugene). , and fluorescence observation was performed using a Fluorescent microscope (BX60; Olympus, Japan). An example of the results is shown in FIG.
図１１に示される通り、被験物質投与により認知能の改善が認められた個体では、コントロール群に比べて脳アミロイド斑の数及び表面積が低下していた。 As shown in FIG. 11, in individuals whose cognitive abilities were improved by administration of the test substance, the number and surface area of brain amyloid plaques were reduced compared to the control group.
（３）大脳皮質におけるL-PGDS発現に対する評価（免疫組織化学染色法）
上記（１）で作製した被験物質投与群及びコントロール群の冠状面切片について、抗L-PGDS抗体（anti-prostaglandin D synthase (lipocalin) antibody [EP12357]、Abcam社、1：1000）による免疫組織化学的染色を行った。L-PGDSはavidin-biotin horseradish peroxidase（HRP）complex キット（Vector Laboratories社）と3,3'-diaminobenzidine tetrahydrochloride（DAB）反応を用いて検出した。結果の一例を図１２に示す。
(3) Evaluation of L-PGDS expression in the cerebral cortex (immunohistochemical staining method)
Immunohistochemistry using anti-L-PGDS antibody (anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1:1000) of the coronal sections of the test substance administration group and control group prepared in (1) above. Target staining was performed. L-PGDS was detected using an avidin-biotin horseradish peroxidase (HRP) complex kit (Vector Laboratories) and a 3,3'-diaminobenzidine tetrahydrochloride (DAB) reaction. An example of the results is shown in FIG. 12.
図１２に示される通り、被験物質投与群の脳では血管膣近傍にL-PGDSの特異染色が認められた（パネルBの矢印部分）のに対し、コントロール群の脳ではL-PGDSの特異染色は認められなかった（パネルA）。 As shown in Figure 12, specific staining of L-PGDS was observed near the blood vessel vagina in the brains of the test substance administration group (arrow area in panel B), whereas specific staining of L-PGDS was observed in the brains of the control group. was not observed (Panel A).
また、上記（１）で作製した被験物質投与群の冠状面切片について、L-PGDS（anti-prostaglandin D synthase (lipocalin) antibody [EP12357]、Abcam社、1:1000）及び血管内皮細胞マーカーCD31（anti-mouse CD31 (PECAM-1) monoclonal antibody、550274、BD Pharmingen社、1:1000）に対する特異的抗体を用いた免疫組織化学的検討を行った。上記の一次抗体を結合させた切片において、Alexa Fluor 488-conjugated抗体又はAlexa Fluor 555-conjugated抗体（1:500；Molecular Probes、Eugene）と反応後に、4',6-diamidino-2-phenylindole（DAPI；1:000；Kirkegaard & Perry Laboratories社）による核染色を行い、confocal laser microscope （LSM780；Carl Zeiss、Jena, Germany）で蛍光観察した。多重染色法により、L-PGDS（パネルB）とCD31（パネルC）の画像を重ね合わせて、L-PGDS とCD31の分布について比較した（パネルA）。結果の一例を図１３に示す。 In addition, for the coronal section of the test substance administration group prepared in (1) above, L-PGDS (anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1:1000) and vascular endothelial cell marker CD31 ( Immunohistochemical studies were performed using a specific antibody against anti-mouse CD31 (PECAM-1) monoclonal antibody, 550274, BD Pharmingen, 1:1000). After reacting with Alexa Fluor 488-conjugated antibody or Alexa Fluor 555-conjugated antibody (1:500; Molecular Probes, Eugene) on the sections bound with the above primary antibody, 4',6-diamidino-2-phenylindole (DAPI ; 1:000; Nuclear staining was performed using Kirkegaard & Perry Laboratories), and fluorescence observation was performed using a confocal laser microscope (LSM780; Carl Zeiss, Jena, Germany). Images of L-PGDS (panel B) and CD31 (panel C) were superimposed using multiple staining, and the distributions of L-PGDS and CD31 were compared (panel A). An example of the results is shown in FIG.
図１３に示される通り、被験物質投与群の脳では、L-PGDS（緑色蛍光）が血管内皮細胞マーカーCD31（赤色蛍光）の周囲に分布していることが認められた。これより、被験物質の投与に応答して、血管内皮細胞を覆うように存在するペリサイト（又はiSC）においてL-PGDSの発現が上昇したと考えられた。 As shown in FIG. 13, in the brains of the test substance administration group, L-PGDS (green fluorescence) was observed to be distributed around the vascular endothelial cell marker CD31 (red fluorescence). From this, it was considered that in response to administration of the test substance, the expression of L-PGDS increased in pericytes (or iSCs) that were present covering vascular endothelial cells.
（４）脳内アミロイドβ沈着とL-PGDS発現に対する評価（ウエスタンブロッティング法）
上記（１）で採取後急速凍結した左半球脳をPotter型ホモジェナイザーによりホモジナイズして抽出したタンパクをSDS-PAGE（BIO-RAD Any kD（商標））で分離し、PVDF膜（Immun-Blot PVDF メンブレン、BIO-RAD社）に転写した。Bloking One（nacalai tesque社）でブロッキング後、特異的な抗体を用いたウエスタンブロッティング法により、アミロイドβ（anti-β-amyloid、1-16、[SIG-39300]、BioLegend社、1:1000）及びL-PGDS（anti-Prostaglandin D Synthase（Lipocalin）antibody [EP12357]、Abcam社、1:2000）を検出した。検出には高感度化学発光法（Chemi-Lumi One L, nacalai tesque 社）を使用した。結果の一例を図１４に示す。
(4) Evaluation of intracerebral amyloid β deposition and L-PGDS expression (Western blotting method)
The left hemisphere brain was rapidly frozen after collection in (1) above and was homogenized using a Potter homogenizer. The extracted proteins were separated using SDS-PAGE (BIO-RAD Any kD (trademark)), and the proteins were separated using a PVDF membrane (Immun-Blot). Transferred to PVDF membrane (BIO-RAD). After blocking with Blocking One (Nacalai Tesque), amyloid β (anti-β-amyloid, 1-16, [SIG-39300], BioLegend, 1:1000) and L-PGDS (anti-Prostaglandin D Synthase (Lipocalin) antibody [EP12357], Abcam, 1:2000) was detected. A highly sensitive chemiluminescence method (Chemi-Lumi One L, Nacalai Tesque) was used for detection. An example of the results is shown in FIG.
図１４のパネルA※印部分に示される通り、脳内アミロイドβ量については、被験物質投与により認知能の改善が認められた被験物質投与群のマウス（個体番号4、29）ではコントロール群のマウス（個体番号14、30）に比べて減少していることが認められた。この結果は、試験例7（２）の免疫組織化学染色法（図１１）において、コントロール群に対して被験物質投与では脳内アミロイドβ抗体陽性産物の数及び表面積サイズが抑制されていた結果と相関するものであった。また、図１４のパネルB＊印部分に示される通り、被験物質投与群では脳内L-PGDS量がコントロール群に比べて増加していることから、被験物質が脳内L-PGDS産生を亢進していることが認められた。上記試験例7の一連の結果から、被験物質がL-PGDSの脳内発現量を増加させることにより、脳内アミロイドβの沈着を阻害し、引いては認知機能を改善させる可能性が考えられた。
As shown in panel A* of Figure 14, the amount of amyloid β in the brain was found to be higher in mice in the test substance administration group (
試験例８：ヒト由来ペリサイトにおけるL-PGDS発現促進要因の検討（古典的RT-PCR法）
上述したとおり、被験物質に対する応答性が認められたiSCは脳ペリサイトに由来すると考えられていることから、市販のヒト由来ペリサイト株Human brain vascular pericytes（ScienCell社）において、異なる酸素及び／又はグルコース濃度の条件下で、被験物質のL-PGDS発現能を調べた。具体的には、0、50、500 mNU/mLの各用量の被験物質存在下、各々、（1）iSC が反応性を示す条件（4.5g/L Glucose及び20％ O2）、（2）低グルコース条件（90mg/L Glucose及び20％ O2）並びに（3）低酸素・低グルコース条件（90mg/L Glucose及び1％ O2）で、iSCを4日間培養（FBS不含 DMEM培地 F/-/-）した。RNeasy Mini Kit（QIAGEN社）により回収された全RNAに対して、SuperScript III One-Step RT-PCR System with Platinum（Invitrogen社）を用いた古典的RT-PCR法によってL-PGDS遺伝子（PTGDS）の発現量を半定量した（35サイクル）。PCR産物を2％アガロースゲル電気泳動により分離し、PTGDS及びβ-Actinのバンドを臭化エチジウムで染色して可視的に検出した。なお、PTGDS及びβ-Actin用プライマーは、上記試験例２－１で用いたものと同じものを使用した。結果の一例を図１５に示す。
Test Example 8: Examination of factors promoting L-PGDS expression in human-derived pericytes (classical RT-PCR method)
As mentioned above, iSCs that were found to be responsive to the test substance are thought to be derived from brain pericytes. The ability of the test substance to express L-PGDS was examined under conditions of glucose concentration. Specifically, in the presence of the test substance at each dose of 0, 50, and 500 mNU/mL, (1) conditions under which iSCs show reactivity (4.5 g/L Glucose and 20% O 2 ), (2) iSCs were cultured for 4 days under low glucose conditions (90 mg/L Glucose and 20% O 2 ) and (3) hypoxia/low glucose conditions (90 mg/L Glucose and 1% O 2 ) (FBS-free DMEM medium F/ -/-)did. L-PGDS gene (PTGDS) was isolated by classical RT-PCR using SuperScript III One-Step RT-PCR System with Platinum (Invitrogen) on total RNA recovered with RNeasy Mini Kit (QIAGEN). Expression levels were semi-quantified (35 cycles). PCR products were separated by 2% agarose gel electrophoresis, and PTGDS and β-Actin bands were visually detected by staining with ethidium bromide. The primers for PTGDS and β-Actin were the same as those used in Test Example 2-1 above. An example of the results is shown in FIG. 15.
図１５に示される通り、条件（1）で培養した細胞のL-PGDS遺伝子の転写は検出可能ではあったものの、被験物質に対する応答性は観察されなかった。一方、低酸素（1%）かつ低グルコース濃度（90 mg/L）である条件（3）で培養した細胞においては、被験物質に対する顕著な応答性が観察された。低酸素・低グルコース条件は脳内の虚血状態を模倣すると考えられるため、このような病態時にペリサイト（あるいはiSC）が外界の刺激に対する応答性を獲得し、L-PGDSのような細胞保護性のタンパクを遊離するものと考えられた。 As shown in FIG. 15, although transcription of the L-PGDS gene in cells cultured under condition (1) was detectable, no responsiveness to the test substance was observed. On the other hand, remarkable responsiveness to the test substance was observed in cells cultured under conditions (3) of hypoxia (1%) and low glucose concentration (90 mg/L). Since hypoxic and low glucose conditions are thought to mimic ischemic conditions in the brain, pericytes (or iSCs) acquire responsiveness to external stimuli during such pathological conditions, and provide cell protection such as L-PGDS. It was thought that it liberates the sex protein.
L-PGDSは主として脳内に発現し、様々な疎水性低分子の結合・輸送及びスカベンジャーとしての役割を担って、脳内環境調整・脳保護機能や睡眠調節機能等の様々な機能を有するタンパク質であるとされていることから、本発明L-PGDS産生促進剤は、脳梗塞等の脳血管障害、アルツハイマー病等の認知症、不眠症等のL-PGDSが関与する疾患の予防・治療又は再発予防薬として有用である。特に、本抽出物及び本抽出物を含有する製剤は優れたL-PGDS産生促進剤であると共に副作用等の問題点の少ない安全性の高い薬剤として、有用性の高いものである。また、ペリサイト又はペリサイトから脱分化したiSCにおけるL-PGDS産生促進作用を指標として、L-PGDSが関与する疾患の予防・治療又は再発予防に有用な物質、特に脳保護作用あるいは睡眠作用を有する物質の本発明スクリーニング方法は、新しい治療薬の開発に寄与する大変有用な方法である。 L-PGDS is a protein that is mainly expressed in the brain and plays the role of binding and transporting various hydrophobic small molecules and as a scavenger, and has various functions such as regulating the brain environment, protecting the brain, and regulating sleep. Therefore, the L-PGDS production promoter of the present invention can be used for the prevention and treatment of diseases involving L-PGDS, such as cerebrovascular disorders such as cerebral infarction, dementia such as Alzheimer's disease, and insomnia. It is useful as a relapse prevention drug. In particular, the present extract and preparations containing the present extract are highly useful as not only excellent L-PGDS production promoters but also highly safe drugs with few problems such as side effects. In addition, using the L-PGDS production promoting effect in pericytes or iSCs dedifferentiated from pericytes as an indicator, we will use substances useful for the prevention, treatment, or recurrence prevention of diseases related to L-PGDS, especially brain protective effects or sleep effects. The screening method of the present invention for substances possessing the above-mentioned properties is a very useful method that contributes to the development of new therapeutic agents.
Claims (3)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017253170 | 2017-12-28 | ||
| JP2017253170 | 2017-12-28 | ||
| JP2019530517A JP6607655B1 (en) | 2017-12-28 | 2018-12-27 | Lipocalin-type prostaglandin D2 synthase production promoter |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019530517A Division JP6607655B1 (en) | 2017-12-28 | 2018-12-27 | Lipocalin-type prostaglandin D2 synthase production promoter |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2020007376A JP2020007376A (en) | 2020-01-16 |
| JP7411175B2 true JP7411175B2 (en) | 2024-01-11 |
Family
ID=67067534
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019530517A Active JP6607655B1 (en) | 2017-12-28 | 2018-12-27 | Lipocalin-type prostaglandin D2 synthase production promoter |
| JP2019190666A Active JP7411175B2 (en) | 2017-12-28 | 2019-10-18 | Lipocalin-type prostaglandin D2 synthase production promoter |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019530517A Active JP6607655B1 (en) | 2017-12-28 | 2018-12-27 | Lipocalin-type prostaglandin D2 synthase production promoter |
Country Status (9)
| Country | Link |
|---|---|
| US (2) | US20200338135A1 (en) |
| EP (1) | EP3733205A4 (en) |
| JP (2) | JP6607655B1 (en) |
| KR (1) | KR20200103647A (en) |
| CN (2) | CN111511403A (en) |
| AU (1) | AU2018394182A1 (en) |
| CA (1) | CA3087018A1 (en) |
| TW (1) | TWI814760B (en) |
| WO (1) | WO2019131879A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3128060A1 (en) * | 2019-01-30 | 2020-08-06 | Nippon Zoki Pharmaceutical Co., Ltd. | Inhibiting or alleviating agent for inflammation in the brain |
| WO2023184470A1 (en) * | 2022-04-01 | 2023-10-05 | 星相生物技术有限公司 | Use of extracts from rabbit skin inflamed by vaccinia virus in treatment of alzheimer's disease |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000016942A (en) | 1998-04-27 | 2000-01-18 | Nippon Zoki Pharmaceut Co Ltd | Therapeutic agent for ischemic disease |
| JP2001103869A (en) | 1999-10-06 | 2001-04-17 | Japan Science & Technology Corp | Lipocalin-type prostaglandin d synthetase transgenic animal and examination using the animal |
| JP2007284351A (en) | 2004-07-27 | 2007-11-01 | Osaka Bioscience Institute | Substance inhibiting aggregation of amyloid protein and action thereof |
| JP2012508192A (en) | 2008-11-11 | 2012-04-05 | バンワールド ファーマシューティカル（ルガオ） カンパニー リミテッド | Use of an extract from rabbit skin inflamed by vaccinia virus in the manufacture of a medicament for the treatment of acute cerebrovascular disease |
| JP2014076961A (en) | 2012-10-10 | 2014-05-01 | Nippon Zoki Pharmaceut Co Ltd | Extract and preparation |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6479657A (en) * | 1987-09-22 | 1989-03-24 | Nippon Zoki Pharmaceutical Co | Vital decision of medicine |
| AU2729400A (en) * | 1999-01-19 | 2000-08-07 | James Daly | Application of a viral complement inhibitory protein in the treatment and diagnosis of alzheimer's disease |
| WO2002008756A1 (en) * | 2000-07-21 | 2002-01-31 | Maruha Corporation | Method of differentiating dementing diseases |
| JP3818657B2 (en) | 2004-12-01 | 2006-09-06 | 日本臓器製薬株式会社 | Dried product and production method thereof |
| CN100542543C (en) | 2005-06-17 | 2009-09-23 | 日本脏器制药株式会社 | Dry thing and autofrettage thereof |
| WO2009039476A1 (en) * | 2007-09-20 | 2009-03-26 | Johns Hopkins University | Treatment and prevention of ischemic brain injury |
| EP2587265B1 (en) | 2010-06-25 | 2016-05-04 | Nippon Zoki Pharmaceutical Co., Ltd. | Method for determination or evaluation of test substance |
| US9835621B2 (en) * | 2011-07-14 | 2017-12-05 | University Of Kentucky Research Foundation | Process for detection of alzheimer's disease from a serum sample |
| CN115629169A (en) * | 2013-04-30 | 2023-01-20 | 日本脏器制药株式会社 | Extract and preparation containing the same |
| KR20190121782A (en) * | 2017-03-06 | 2019-10-28 | 준 리우 | Inhibitors or Mitigators for Aβ-Induced Damage |
-
2018
- 2018-12-27 KR KR1020207015608A patent/KR20200103647A/en unknown
- 2018-12-27 WO PCT/JP2018/048141 patent/WO2019131879A1/en unknown
- 2018-12-27 US US16/958,962 patent/US20200338135A1/en not_active Abandoned
- 2018-12-27 JP JP2019530517A patent/JP6607655B1/en active Active
- 2018-12-27 EP EP18895831.8A patent/EP3733205A4/en active Pending
- 2018-12-27 CN CN201880084260.8A patent/CN111511403A/en active Pending
- 2018-12-27 CN CN202310272473.6A patent/CN116270743A/en active Pending
- 2018-12-27 AU AU2018394182A patent/AU2018394182A1/en active Pending
- 2018-12-27 CA CA3087018A patent/CA3087018A1/en active Pending
- 2018-12-28 TW TW107147748A patent/TWI814760B/en active
-
2019
- 2019-10-18 JP JP2019190666A patent/JP7411175B2/en active Active
-
2023
- 2023-07-20 US US18/355,925 patent/US20240000853A1/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000016942A (en) | 1998-04-27 | 2000-01-18 | Nippon Zoki Pharmaceut Co Ltd | Therapeutic agent for ischemic disease |
| JP2001103869A (en) | 1999-10-06 | 2001-04-17 | Japan Science & Technology Corp | Lipocalin-type prostaglandin d synthetase transgenic animal and examination using the animal |
| JP2007284351A (en) | 2004-07-27 | 2007-11-01 | Osaka Bioscience Institute | Substance inhibiting aggregation of amyloid protein and action thereof |
| JP2012508192A (en) | 2008-11-11 | 2012-04-05 | バンワールド ファーマシューティカル（ルガオ） カンパニー リミテッド | Use of an extract from rabbit skin inflamed by vaccinia virus in the manufacture of a medicament for the treatment of acute cerebrovascular disease |
| JP2014076961A (en) | 2012-10-10 | 2014-05-01 | Nippon Zoki Pharmaceut Co Ltd | Extract and preparation |
Non-Patent Citations (6)
| Title |
|---|
| GARCIA-FERNANDEZ LF et al.，Dexamethasone induces lipocalin-type prostaglandin D synthase gene expression in mouse neuronal cells，Journal of Neurochemistry，2000年，Vol.75, No.2，p.460-470，ISSN 0022-3042 |
| KANEKIYO T et al.，Lipocalin-type prostaglandin D synthase/beta-trace is a major amyloid beta-chaperone in human cerebrospinal fluid，Proceedings of the National Academy of Sciences of the United States of America，2007年04月10日，Vol.104, No.15，p.6412-6417，ISSN 0027-8424 |
| SALEEM S et al.，Lipocalin-prostaglandin D synthase is a critical beneficial factor in transient and permanent focal cerebral ischemia，Neuroscience，2009年，Vol.160, No.1，p.248-254，ISSN 0306-4522 |
| 木村 宏 ほか，老年期の痴呆症状を呈する患者に対するノイロトロピン(R)の使用経験 ―パイロットスタディー―，薬理と治療，1987年10月，第15巻, 第10号，p.4235-4251，ISSN 0386-3603 |
| 松山 知弘, 中込 隆之，脳ペリサイトをめぐる脳保護と再生，脳循環代謝，2015年，第26巻, 第2号，p.145-149，ISSN 0915-9401 |
| 永田 奈々恵, 裏出 良博，睡眠物質プロスタグランジンD2，ねむりと医療，2011年，第4巻, 第2号，p.65-69，ISSN 1883-0552 |
Also Published As
| Publication number | Publication date |
|---|---|
| US20240000853A1 (en) | 2024-01-04 |
| EP3733205A4 (en) | 2021-12-08 |
| JPWO2019131879A1 (en) | 2020-01-16 |
| KR20200103647A (en) | 2020-09-02 |
| CN116270743A (en) | 2023-06-23 |
| TW201932130A (en) | 2019-08-16 |
| TWI814760B (en) | 2023-09-11 |
| US20200338135A1 (en) | 2020-10-29 |
| AU2018394182A1 (en) | 2020-07-16 |
| JP2020007376A (en) | 2020-01-16 |
| EP3733205A1 (en) | 2020-11-04 |
| CN111511403A (en) | 2020-08-07 |
| JP6607655B1 (en) | 2019-11-20 |
| CA3087018A1 (en) | 2019-07-04 |
| WO2019131879A1 (en) | 2019-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Regulatory role of NADPH oxidase 2 in the polarization dynamics and neurotoxicity of microglia/macrophages after traumatic brain injury | |
| JP5712207B2 (en) | Compositions and methods for prevention and treatment of brain diseases and conditions | |
| US20240000853A1 (en) | Lipocalin-type prostaglandin d2 synthase production promoting agent | |
| Zhang et al. | Valproate promotes survival of retinal ganglion cells in a rat model of optic nerve crush | |
| US20200061101A1 (en) | Compositions prepared from poultry and methods of their use | |
| EP2587265B1 (en) | Method for determination or evaluation of test substance | |
| Zhang et al. | Carbon monoxide-releasing molecule-3 protects against cortical pyroptosis induced by hemorrhagic shock and resuscitation via mitochondrial regulation | |
| Chen et al. | Interleukin-33 reduces neuronal damage and white matter injury via selective microglia M2 polarization after intracerebral hemorrhage in rats | |
| Katnik et al. | Treatment with afobazole at delayed time points following ischemic stroke improves long-term functional and histological outcomes | |
| Hong et al. | Anti-inflammatory and anti-arthritic effects of the ethanolic extract of Aralia continentalis Kitag. in IL-1β-stimulated human fibroblast-like synoviocytes and rodent models of polyarthritis and nociception | |
| Hou et al. | Acute spinal cord injury in rats should target activated autophagy | |
| Chen et al. | Tert-butylhydroquinone enhanced angiogenesis and astrocyte activation by activating nuclear factor-E2-related factor 2/heme oxygenase-1 after focal cerebral ischemia in mice | |
| Hashimoto et al. | Long-term activation of c-Fos and c-Jun in optic nerve head astrocytes in experimental ocular hypertension in monkeys and after exposure to elevated pressure in vitro | |
| Zhang et al. | Thymosin beta4 promotes oligodendrogenesis in the demyelinating central nervous system | |
| Yan et al. | Endogenous BMP-4/ROS/COX-2 mediated IPC and resveratrol alleviated brain damage | |
| Lee et al. | Rg3-enriched Korean Red Ginseng extract inhibits blood-brain barrier disruption in an animal model of multiple sclerosis by modulating expression of NADPH oxidase 2 and 4 | |
| Wang et al. | Effects of JIP3 on epileptic seizures: evidence from temporal lobe epilepsy patients, kainic-induced acute seizures and pentylenetetrazole-induced kindled seizures | |
| Huang et al. | Targeting integrated stress response regulates microglial M1/M2 polarization and attenuates neuroinflammation following surgical brain injury in rat | |
| Zhang et al. | Recombinant osteopontin provides protection for cerebral infarction by inhibiting the NLRP3 inflammasome in microglia | |
| WO2020154941A1 (en) | Inhibiting or alleviating agent for inflammation in the brain | |
| Gong et al. | Baicalein promotes the microglia M2 polarization and suppresses apoptosis by targeting HMOX1/PDE4D to alleviate Alzheimer’s disease | |
| KR100744015B1 (en) | Health drink for dementia prevention using a chinese medicine | |
| Gao et al. | Spatial–temporal expression of NDRG2 in brain tissues in a rat model of intracerebral hemorrhage: A pilot study | |
| JP2022541720A (en) | Production and use of ENAMPT contained in extracellular vesicles | |
| Zhou et al. | CircDYM attenuates microglial apoptosis via CEBPB/ZC3H4 axis in LPS-induced mouse model of depression |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20211115 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20221108 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20221215 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230307 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20230613 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230912 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20231030 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20231205 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20231213 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7411175 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |